U.S. patent number 4,424,343 [Application Number 06/203,098] was granted by the patent office on 1984-01-03 for preparation of 1-n-[.omega.-amino-.alpha.-hydroxyalkanoyl]kanamycin polysilylates and products.
This patent grant is currently assigned to Bristol Myers Company. Invention is credited to Martin J. Cron, John G. Keil, Jeng S. Lin, Mariano V. Ruggeri, Derek Walker.
United States Patent |
4,424,343 |
Cron , et al. |
* January 3, 1984 |
**Please see images for:
( Certificate of Correction ) ** |
Preparation of 1-N-[.omega.-amino-.alpha.-hydroxyalkanoyl]kanamycin
polysilylates and products
Abstract
An improved process for the preparation of
1-N-[.omega.-amino-.alpha.-hydroxyalkanoyl]kanamycins comprises
acylating a polysilylated kanamycin in a substantially anhydrous
organic solvent with an acylating derivative of an
.omega.-amino-.alpha.-hydroxyalkanoic acid.
Inventors: |
Cron; Martin J. (Fayetteville,
NY), Keil; John G. (Manlius, NY), Lin; Jeng S. (Clay,
NY), Ruggeri; Mariano V. (Liverpool, NY), Walker;
Derek (Jamesville, NY) |
Assignee: |
Bristol Myers Company (New
York, NY)
|
[*] Notice: |
The portion of the term of this patent
subsequent to August 31, 1999 has been disclaimed. |
Family
ID: |
26898311 |
Appl.
No.: |
06/203,098 |
Filed: |
November 3, 1980 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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8730 |
Feb 2, 1979 |
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888585 |
Mar 20, 1978 |
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896430 |
Apr 14, 1978 |
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791806 |
Apr 28, 1977 |
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Current U.S.
Class: |
536/13.8;
536/13.7 |
Current CPC
Class: |
C07H
15/234 (20130101) |
Current International
Class: |
C07H
15/00 (20060101); C07H 15/22 (20060101); C07H
015/22 () |
Field of
Search: |
;536/10,17R,13.7,13.8 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Murata et al., "Chem. Abst.", vol. 79, 1973, p. 83498(y)..
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Primary Examiner: Brown; Johnnie R.
Attorney, Agent or Firm: Lloyd; Richard R.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATION
This application is a continuation of our prior co-pending
application Ser. No. 8,730, filed Feb. 2, 1979, which is a
continuation-in-part of our prior, co-pending applications Ser. No.
888,585, filed Mar. 20, 1978, and Ser. No. 896,430, filed Apr. 14,
1978, each of which is a continuation-in-part of our prior,
co-pending application Ser. No. 791,806, filed Apr. 28, 1977, and
all now abandoned.
Claims
We claim:
1. The process for the preparation of a
1-N-[.omega.-amino-.alpha.-hydroxyalkanoyl]kanamycin A or B having
the formula ##STR30## in which R is OH or NH.sub.2 and n is an
integer of from 0 to 2, or a nontoxic pharmaceutically acceptable
acid addition salt thereof, which comprises acylating polysilylated
kanamycin A or B or polysilylated kanamycin A or B containing a
conventional non-silyl amino-blocking group on the 3-amino group,
the 6'-amino group or the 3-amino and 6'-amino groups, with an
acylating derivative of the acid of the formula ##STR31## in which
n is an integer of from 0 to 2 and B is a conventional
amino-blocking group, in a substantially anhydrous organic solvent,
and subsequently removing all blocking groups by conventional
means; wherein the silyl moieties on the polysilylated kanamycin A
or B are selected from trimethylsilyl, triethylsilyl,
tri-n-propylsilyl, methyldichlorosilyl, dimethylchlorosilyl,
methyldiethylsilyl, dimethylethylsilyl, dimethyl-t-butylsilyl,
phenyldimethylsilyl, benzylmethylethylsilyl,
phenylmethylethylsilyl, triphenylsilyl, tri-o-tolylsilyl,
tri-p-tolylsilyl and tri-p-dimethylaminophenylsilyl, and wherein
the amino-blocking groups on the starting polysilylated kanamycin A
or B and on the acylating derivative of the acid are independently
selected from those of the formulae ##STR32## wherein R.sup.1 and
R.sup.2 are alike or different and each is H, F, Cl, Br, NO.sub.2,
OH, (lower)alkyl or (lower)alkoxy, X is Cl, Br, F or I and Y is H,
Cl, Br, F or I.
2. The process for the preparation of a
1-N-[L-(-)-.omega.-amino-.alpha.-hydroxyalkanoyl]kanamycin A having
the formula ##STR33## in which n is an integer of from 0 to 2, or a
nontoxic pharmaceutically acceptable acid addition salt thereof,
which comprises acylating polysilylated kanamycin A with an
acylating derivative of the acid of the formula ##STR34## in which
n is an integer of from 0 to 2 and B is a conventional
amino-blocking group, in a substantially anhydrous organic solvent,
and subsequently removing all blocking groups by conventional
means; wherein the silyl moieties on the polysilylated kanamycin A
are selected from trimethylsilyl, triethylsilyl, tri-n-propylsilyl,
methyldichlorosilyl, dimethylchlorosilyl, methyldiethylsilyl,
dimethylethylsilyl, dimethyl-t-butylsilyl, phenyldimethylsilyl,
benzylmethylethylsilyl, phenylmethylethylsilyl, triphenylsilyl,
tri-o-tolylsilyl, tri-p-tolylsilyl and
tri-p-dimethylaminophenylsilyl, and the amino-blocking group on the
acylating derivative of the acid is selected from those of the
formulae ##STR35## wherein R.sup.1 and R.sup.2 are alike or
different and each is H, F, Cl, Br, NO.sub.2, OH, (lower)alkyl or
(lower)alkoxy, X is Cl, Br, F or I and Y is H, Cl, Br, F or I.
3. The process of claim 2 wherein the acylating derivative of the
acid is an active ester or a mixed acid anhydride.
4. A process of claim 3 in which the polysilylated kanamycin A
starting material contains an average number of silyl groups per
molecule of from 4 to 8, n is 1 and the silyl groups are
trimethylsilyl.
5. The process of claim 3 wherein the polysilylated kanamycin A
contains a conventional non-silyl amino-blocking group selected
from those of the formulae ##STR36## wherein R.sup.1 and R.sup.2
are alike or different and each is H, F, Cl, Br, NO.sub.2, OH,
(lower)alkyl or (lower)alkoxy, X is Cl, Br, F or I and Y is H, Cl,
Br, F or I on the 6'-amino group.
6. A process of claim 5 in which the polysilylated kanamycin A
starting material contains an average number of silyl groups per
molecule of from 3 to 7.
7. A process of claim 6 in which the amino-blocking group on the
6'-amino group of the polysilylated kanamycin A is the
carbobenzyloxy group, n is 1 and the silyl groups are
trimethylsilyl.
8. The process of claim 3 wherein the polysilylated kanamycin A
contains conventional non-silyl amino-blocking groups selected from
those of the formulae ##STR37## wherein R.sup.1 and R.sup.2 are
alike or different and each is H, F, Cl, Br, NO.sub.2, OH,
(lower)alkyl or (lower)alkoxy, X is Cl, Br, F or I and Y is H, Cl,
Br, F or I on the 3-amino and 6'-amino groups.
9. A process of claim 8 in which the polysilylated kanamycin A
starting material contains an average number of silyl groups per
molecule of from 3 to 6.
10. A process of claim 9 in which the amino-blocking groups on the
3-amino and 6'-amino groups of the polysilylated kanamycin A are
carbobenzyloxy groups, n is 1 and the silyl groups are
trimethylsilyl.
11. The process for the preparation of a
1-N-[L-(-)-.omega.-amino-.alpha.-hydroxyalkanoyl]kanamycin B having
the formula ##STR38## in which n is an integer of from 0 to 2, or a
nontoxic pharmaceutically acceptable acid addition salt thereof,
which comprises acylating polysilylated kanamycin B with an
acylating derivative of the acid of the formula ##STR39## in which
n is an integer of from 0 to 2 and B is a conventional
amino-blocking group, in a substantially anhydrous organic solvent,
and subsequently removing all blocking groups by conventional
means; wherein the silyl moieties on the polysilylated kanamycin B
are selected from trimethylsilyl, triethylsilyl, tri-n-propylsilyl,
methyldichlorosilyl, dimethylchlorosilyl, methyldiethylsilyl,
dimethylethylsilyl, dimethyl-t-butylsilyl, phenyldimethylsilyl,
benzylmethylethylsilyl, phenylmethylethylsilyl, triphenylsilyl,
tri-o-tolylsilyl, tri-p-tolylsilyl and
tri-p-dimethylaminophenylsilyl, and the amino blocking group on the
acylating derivative of the acid is selected from those of the
formulae ##STR40## wherein R.sup.1 and R.sup.2 are alike or
different and each is H, F, Cl, Br, NO.sub.2, OH, (lower)alkyl or
(lower)alkoxy, X is Cl, Br, F or I and Y is H, Cl, Br, F or I.
12. The process of claim 11 wherein the polysilylated kanamycin B
contains a conventional non-silyl amino-blocking group selected
from those of the formulae ##STR41## wherein R.sup.1 and R.sup.2
are alike or different and each is H, F, Cl, Br, NO.sub.2, OH,
(lower)alkyl or (lower)alkoxy, X is Cl, Br, F or I and Y is H, Cl,
Br, F or I on the 6'-amino group.
13. The process of claim 11 wherein the polysilylated kanamycin B
contains conventional non-silyl amino-blocking groups selected from
those of the formulae ##STR42## wherein R.sup.1 and R.sup.2 are
alike or different and each is H, F, Cl, Br, NO.sub.2, OH,
(lower)alkyl or (lower)alkoxy, X is Cl, Br, F or I and Y is H, Cl,
Br, F or I on the 3-amino and 6'-amino groups.
14. Polysilylated kanamycin A or B or polysilylated kanamycin A or
B containing a conventional non-silyl amino-blocking group on the
3-amino, 6'-amino or 3-amino and 6'-amino groups; wherein the silyl
moieties on the polysilylated kanamycin A or B are selected from
trimethylsilyl, triethylsilyl, tri-n-propylsilyl,
methyldichlorosilyl, dimethylchlorosilyl, methyldiethylsilyl,
dimethylethylsilyl, dimethyl-t-butylsilyl, phenyldimethylsilyl,
benzylmethylethylsilyl, phenylmethylethylsilyl, triphenylsilyl,
tri-o-tolylsilyl, tri-p-tolylsilyl and
tri-p-dimethylaminophenylsilyl, and the amino-blocking groups are
selected from those of the formulae ##STR43## wherein R.sup.1 and
R.sup.2 are alike or different and each is H, F, Cl, Br, NO.sub.2,
OH, (lower)alkyl or (lower)alkoxy, X is Cl, Br, F or I and Y is H,
Cl, Br, F or I.
15. A polysilylated kanamycin A of claim 14 containing an average
number of silyl groups per molecule of from 3 to 7.
16. A polysilylated kanamycin A of claim 15 in which the non-silyl
amino-blocking group is the carbobenzyloxy group and the silyl
groups are trimethylsilyl.
17. A polysilylated kanamycin A of claim 14 containing an average
number of silyl groups per molecule of from 3 to 6.
18. A polysilylated kanamycin A of claim 17 in which the non-silyl
amino-blocking groups are carbobenzyloxy groups and the silyl
groups are trimethylsilyl.
19. Polysilylated kanamycin B having a conventional non-silyl
amino-blocking group, as defined in claim 14, on the 6'-amino group
and containing an average number of silyl groups, as defined in
claim 14, per molecule of from 3 to 7.
20. Polysilylated kanamycin B having conventional non-silyl
amino-blocking groups, as defined in claim 14, on the 3-amino and
6'-amino groups, and containing an average number of silyl groups,
as defined in claim 14, per molecule of from 3 to 6.
21. A polysilylated kanamycin B of claim 9 or 20 in which the
non-silyl amino-blocking group is the carbobenzyloxy group and the
silyl groups are trimethylsilyl.
Description
SUMMARY OF THE INVENTION
This invention relates to an improved process for the preparation
of compounds of the formula ##STR1## in which R is OH or NH.sub.2
and n is an integer of from 0 to 2, or a nontoxic pharmaceutically
acceptable acid addition salt thereof, which comprises acylating
polysilylated kanamycin A or B, or polysilylated kanamycin A or B
containing a conventional non-silyl blocking group on the 3-amino
group, the 6'-amino group or the 3-amino and 6'-amino groups, in a
substantially anhydrous organic solvent, with an acylating
derivative of an acid of the formula ##STR2## in which n is an
integer of from 0 to 2 and B is a conventional amino-blocking
group, and subsequently removing all blocking groups by
conventional means.
DESCRIPTION OF THE PRIOR ART
The kanamycins are well-known antibiotics, having been described,
for example in the Merck Index, 8th edition, pp. 597-8. Numerous
derivatives of the kanamycins also are known. The structural
formulae of kanamycins A and B are given below, along with the
standard numbering system used in the art. Hereinafter, where
readily understandable, the various kanamycin derivatives will be
referred to as derivatives of kanamycin A or B rather than by
structural formula, so as to avoid the necessity of comparing
complex structures to determine differences. ##STR3##
U.S. Pat. No. 3,781,268 discloses and claims
1-[L-(-)-.gamma.-amino-.alpha.-hydroxybutyryl]kanamycin A
(amikacin) and B, as well as their mono- and di-carbobenzyloxy
protected derivatives. For lower and higher homologs see U.S. Pat.
Nos. 3,886,139 and 3,904,597. The compounds are prepared by
acylating a 6'-N-protected kanamycin A or B with an acylating
derivative of an N-protected
L-(-)-.gamma.-amino-.alpha.-hydroxybutyric acid, in an aqueous
medium, followed by removal of one or both N-protecting groups.
U.S. Pat. No. 3,974,137 discloses and claims a process for
preparing 1-[L-(-)-.gamma.-amino-.alpha.-hydroxybutyryl]kanamycin A
which comprises reacting 6'-carbobenzyloxykanamycin A with at least
three moles of benzaldehyde, a substituted benzaldehyde or
pivaldehyde, to produce 6'-N-carbobenzyloxykanamycin A containing
Schiff base moieties on the 1,3 and 3"-positions, acylating this
tetra-protected kanamycin A derivative with the
N-hydroxysuccinimide ester of
L-(-)-.gamma.-benzyloxycarbonylamino-.alpha.-hydroxybutyric acid,
and subsequently removing the protecting groups.
Belgian Pat. No. 828192 discloses and claims a process for
preparing 1-[L-(-)-.gamma.-amino-.alpha.-hydroxybutyryl]kanamycin A
by preparation of the same tetra-protected kanamycin A derivative
as in U.S. Pat. No. 3,974,137, acylating with the
N-hydroxy-5-norbornene-2,3-dicarboximide ester of
L-(-)-.gamma.-benzyloxycarbonylamino-.alpha.-hydroxybutyric acid,
and subsequently removing the protecting groups.
U.S. Pat. No. 3,939,143 discloses and claims 1-N-isoseryl(
1-N-.beta.-amino-.alpha.-hydroxypropionyl) derivatives of kanamycin
A, kanamycin B and 3',4'-dideoxykanamycin B, and their preparation
by acylating the appropriate 6'-N-blocked or 2',6'-di-N-blocked
kanamycin with an acylating derivative of an N-protected isoserine,
in an aqueous medium, and subsequently removing the N-protecting
groups.
Trimethylsilyl derivatives of various aminoglycoside antibiotics,
including the kanamycins, are known. They have been prepared to
impart volatility to these relatively non-volatile compounds, for
purposes of gas chromatography and mass spectroscopy analyses.
Bunseki Kagaku, 22, 405-410 (1973), [Chemical Abstracts, 79, 83498y
(1973)] reports gas chromatography and mass spectroscopy analyses
of trimethylsilyl derivatives of kanamycin A, kanamycin B and
neomycin B. These were persilylated compounds in which all hydroxy
and amino groups were silylated.
In J. Am. Chem. Soc., 89, 3364-5 (1967), D. C. DeJongh et al.
report on mass spectrometric structural analysis studies conducted
on the per(N-acetyl)-per(O-trimethylsilyl) derivatives of
paromomycin and paromomycin II.
In Tetrahedron Letters, No. 46, pp 4009-12 (1974), T. Takamoto and
S. Hanessian, report the conversion of paromomycin into a
pseudotrisaccharide by elimination of the diaminohexose unit, and
the confirmation of its structure by high resolution mass
spectroscopic analysis of its per(N-acetyl)-per(O-trimethylsilyl)
derivative. On pages 4013-6 of the same volume, T. Ogawa et al.
report the preparation of a positional isomer of the above
pseudotrisaccharide and confirmation of its structure by high
resolution mass spectroscopy of its
per(N-acetyl)-per(O-trimethylsilyl) derivative.
In The Journal of Antibiotics, 28, 522-9 (1975) P. W. K. Woo
describes the synthesis of 5"-amino-3',4',5"-trideoxybutirosin and
report that its structure is consistent with the mass spectrum of
its penta-N-acetyl-tetrakis-O-trimethylsilyl derivative.
In The Journal of Antibiotics, 26, 374-385 (1973), S. Inouye et al.
report the isolation of a new member of the destomycin group of
aminoglycosides from a culture broth of Streptomyces eurocidicus
SS-56. Its structure was elucidated by means of gas chromatography
of its per(trimethylsilyl) derivative and mass spectroscopy of the
per(O-trimethylsilyl)-N-salicylidene Schiff base derivative.
In The Journal of Antibiotics, 26, 784-6 (1973), M. Kojima and A.
Satoh report the semi-synthesis of several aminoglycoside
antibiotics (e.g. 6'-deamino-6'-hydroxy-1-N-methylkanamycin) by the
addition of deoxystreptamine or neamine analogs to fermentation
broths of deoxystreptamine-negative mutants of Streptomyces
ribosidifucus and Streptomyces kanamyceticus. Their structures were
elucidated by mass spectroscopy of their N-acetyl-O-trimethylsilyl
derivatives.
In Analytical Chemistry, 42, 1661-3 (1970), K. Tsuji and J. H.
Robertson describe a method for the separation and determination of
the kanamycins and paromomycins by silylation and gas
chromatography of the per(trimethylsilyl) derivatives.
In Proc. Nat. Acad. Sci., 63, 198-204 (1969), W. T. Shier et al.
report the preparation of hybrimycins A1, A2, B1 and B2 by
fermentation of a mutant of Streptomyces fradiae, and mass
spectroscopic analysis of their N-acetyl-per(O-trimethylsilyl)
derivatives.
In The Journal of Antibiotics, 26, 790-3 (1973), T. P. Culbertson
et al. report the preparation of 5"-amino-5"-deoxybutirosins A and
B from butirosins A and B. The first steps in the synthesis
involved:
(1) partially N-trifluoroacetylating butirosin base by refluxing in
a mixture of methanol and ethyl trifluoroacetate,
(2) evaporating to dryness, dissolving the residue in pyridine,
treating it with hexamethyldisilizane and trimethylchlorosilane,
then cooling to <10.degree. C. and treating it with
trifluoroacetic anhydride,
(3) evaporating to dryness and hydrolyzing the residue in a 2:1
mixture of ethanol and 2 N acetic acid at reflux, to give
tetra[N-(trifluoroacetyl)]butirosin.
The final products of the synthetic scheme,
5"-amino-5"-deoxybutirosins A and B, also were reacted according to
the above three steps to give
penta[N-(trifluoroacetyl)]-5"-amino-5"-deoxybutirosins A and B.
Although this publication discloses the acylation of a
trimethylsilylated (and partially acylated) aminoglycoside
antibiotic, the result in each instance is complete acylation of
all primary amino groups in the molecule (four in the starting
butirosin and five in the product). The process of the present
invention substantially eliminates polyacylation and provides a
high degree of selectivity of acylation in the desired
1-N-position.
J. J. Wright et al., in The Journal of Antibiotics, 29, 714-719
(1976), describe a general procedure for the selective
1-N-acylation of the gentamicin-sisomicin class of aminoglycosides.
They report that selectivity in the site of acylation is pH
dependent and that the C-1 amino group is the most reactive toward
acylation when the amino groups of the molecule are almost
completely protonated. These conditions are achieved by the
addition of one equivalent of a tertiary amine base to a solution
of the fully neutralized acid addition salt. Although these workers
obtained 1-N-selectivity in the acylation of gentamicin C.sub.1a,
sisomicin and verdamicin, they reported that little selectivity was
observed in the acylation of highly hydroxylated aminoglycosides
such as gentamicin B and kanamycin A.
U.K. Pat. No. 1,460,039 discloses a process for the preparation of
deoxyaminoglycosides, including the kanamycins, by halogenating a
phosphorylated aminoglycoside (one in which the hydroxy group to be
removed has been converted to a phosphonoxy group), to produce the
corresponding aminoglycoside in which the hydroxy group has been
converted to halogen, and reducing the halogen compound to produce
the corresponding deoxyaminoglycoside. Before halogenating the
phosphorylated aminoglycoside, all of its functional groups are
preferably protected by means of silyl or acyl groups.
DETAILED DESCRIPTION
The present invention provides an improved and commercially
attractive process for the preparation of compounds of formula I.
The use of a polysilylated kanamycin A or B as a starting material
gives high solubility in the organic solvent system, thus
permitting reaction at high concentrations. Although the reaction
is usually conducted in solutions containing about 10-20%
polysilylated kanamycin starting material, excellent results have
been obtained at concentrations of about 50% W/V (e.g. 50 gms./100
ml. of solvent).
As with prior art processes, the present process gives a mixture of
acylated products. The desired 1-N-acylated product is separated
from the other products by chromatography and, if desired, the
by-products may be hydrolyzed to the starting kanamycin for
recycling. In prior art processes it was found that any
3"-N-acylated material which was produced caused a loss of about an
equal amount of the desired 1-N-acylated product, due to the great
difficulty of separating the latter from the former. Thus, in
preparing 1-[L-(-)-.gamma.-amino-.alpha.-hydroxybutyryl]kanamycin A
(BB-K8) by various prior art procedures, there is typically also
produced the 3"-N-acylated product (BB-K11), the 3-N-acylated
product (BB-K29), the 6'-N-acylated product (BB-K6) and
polyacylated material, as well as unreacted kanamycin A. Thus, in
commercial production of BB-K8 by acylation of 6'-N-carbobenzyloxy
kanamycin A in an aqueous medium, followed by removal of the
protecting group, we found that about 10% of the desired BB-K8 (2.5
kg. in a 25 kg. batch) usually was lost because of the presence of
BB-K11 as a co-product. A particularly desirable feature of the
present process is the extremely low amount of undesirable
3"-N-acylated product which is produced (typically, none is
detected). When preparing BB-K8 by the present process, BB-K11
(typically is not detected in the reaction mixture.
The present invention provides the process for the preparation of a
1-N-[.omega.-amino-.alpha.-hydroxyalkanoyl]kanamycin A or B having
the formula ##STR4## in which R is OH or NH.sub.2 and n is an
integer of from 0 to 2, or a nontoxic pharmaceutically acceptable
acid addition salt thereof, which comprises acylating polysilylated
kanamycin A or B or polysilylated kanamycin A or B containing a
conventional non-silyl blocking group on the 3-amino group, the
6'-amino group or the 3-amino and 6'-amino groups, with an
acylating derivative of the acid of the formula ##STR5## in which n
is an integer of from 0 to 2 and B is a conventional amino-blocking
group, in a substantially anhydrous organic solvent, and
subsequently removing all blocking groups by conventional
means.
The blocking groups which may be used to protect the 3- and/or
6'-amino groups of the kanamycin and the amino group of the
acylating acid (group B in Formula II) are conventional blocking
groups for the protection of primary amine groups and are well
known to those skilled in the art. Suitable blocking groups include
alkoxycarbonyl groups such as t-butoxycarbonyl and
t-amyloxycarbonyl; aralkoxycarbonyl groups such as
benzyloxycarbonyl; cycloalkyloxycarbonyl groups such as
cyclohexyloxycarbonyl; haloalkoxycarbonyl groups such as
trichloroethoxycarbonyl; acyl groups such as phthaloyl and
o-nitrophenoxyacetyl; haloacetyl groups such as trifluoroacetyl;
and other well-known blocking groups such as 2,4-dinitrophenyl,
trityl, benzyl, alkylbenzyl, etc. Another particularly useful class
of blocking groups are those of the formula RCH.dbd. in which R is
aryl or (lower)alkyl, each of which may be substituted by chloro,
bromo, fluoro, nitro, (lower)alkoxy, or the like. These blocking
groups, which form a Schiff base with the amino group, are
introduced by reaction with the desired aldehyde, e.g. benzaldehyde
or pivaldehyde.
The acylating acid of formula II may be in its (+) or (-) isomeric
form or a mixture of the two isomers (the d,l form), thus producing
the corresponding compound of formula I in which the
1-N-[.omega.-amino-.alpha.-hydroxyalkanoyl] group is in its (+) [or
(R)] form or its (-) [or (S)] form, or a mixture thereof. Each such
isomeric form, and the mixture thereof, is included within the
scope of this invention. In one preferred embodiment, the acylating
acid of formula II is in its (-) form. In another preferred
embodiment the acylating acid of Formula II is in its (+) form.
In one embodiment of the invention the starting material is
polysilylated kanamycin A or B (and preferably polysilylated
kanamycin A). In other embodiments the starting material is
polysilylated kanamycin A or B (and preferably polysilylated
kanamycin A) containing a conventional non-silyl blocking group on
the 3-amino group, the 6'-amino group or the 3-amino and 6'-amino
groups, said blocking group preferably being selected from those of
the formulae ##STR6## wherein R.sup.1 and R.sup.2 are alike or
different and each is H, F, Cl, Br, NO.sub.2, OH, (lower)alkyl or
(lower)alkoxy, and X is Cl, Br, F or I, and Y is H, Cl, Br, F or I.
The most preferred blocking group is the carbobenzyloxy group.
In a preferred embodiment of the invention the acylating derivative
of the acid of Formula II is an active ester, and preferably its
active ester with N-hydroxysuccinimide,
N-hydroxy-5-norbornene-2,3-dicarboximide or N-hydroxyphthalimide.
In another preferred embodiment the acylating derivative of the
acid of Formula II is a mixed acid anhydride, and preferably its
mixed acid anhydride with pivalic acid, benzoic acid,
isobutylcarbonic acid or benzylcarbonic acid.
In another preferred embodiment, amino-blocking group B of the
acylating derivative of the acid of Formula II is selected from
blocking groups of the formulae ##STR7## wherein R.sup.1 and
R.sup.2 are alike or different and each is H, F, Cl, Br, NO.sub.2,
OH, (lower)alkyl or (lower)alkoxy, X is Cl, Br, F or I, and Y is H,
Cl, Br, F or I. The most preferred blocking group is the
carbobenzyloxy group.
In another preferred embodiment the acylating derivative of the
acid of Formula II is an acylating derivative of N-blocked
.gamma.-amino-.alpha.-hydroxybutyric acid. In a more preferred
embodiment the acylating derivative of the acid of Formula II is a
mixed acid anhydride of
.gamma.-benzyloxycarbonylamino-.alpha.-hydroxybutyric acid, and
most preferably its mixed acid anhydride with pivalic acid, benzoic
acid, isobutylcarbonic acid or benzylcarbonic acid. In another more
preferred embodiment, the acylating derivative of the acid of
Formula II in an active ester of .gamma.-benzyloxybutyric acid, and
most preferably its active ester with N-hydroxysuccinimide,
N-hydroxy-5-norbornene-2,3-dicarboximide or
N-hydroxyphthalimide.
In a most preferred embodiment, this invention relates to the
preparation of
1-N-[L-(-)-.gamma.-amino-.alpha.-hydroxybutyryl]kanamycin A or a
nontoxic pharmaceutically acceptable acid addition salt thereof,
which comprises acylating polysilylated kanamycin A with a mixed
acid anhydride of
L-(-)-.gamma.-benzyloxycarbonylamino-.alpha.-hydroxybutyric acid
(and preferably its mixed acid anhydride with pivalic acid, benzoic
acid, isobutylcarbonic acid or benzylcarbonic acid) in a
substantially anhydrous organic solvent, and subsequently removing
all blocking groups.
In another most preferred embodiment, this invention relates to the
preparation of
1-N-[L-(-)-.gamma.-amino-.alpha.-hydroxybutyryl]kanamycin A or a
nontoxic pharmaceutically acceptable acid addition salt thereof,
which comprises acylating polysilylated kanamycin A containing a
carbobenzyloxy group on the 6'-amino moiety with a mixed acid
anhydride of
L-(-)-.gamma.-benzyloxycarbonylamino-.alpha.-hydroxybutyric acid
(and preferably its mixed acid anhydride with pivalic acid, benzoic
acid, isobutylcarbonic acid or benzylcarbonic acid) in a
substantially anhydrous organic solvent, and subsequently removing
all blocking groups.
In another most preferred embodiment, this invention relates to the
preparation of
1-N-[L-(-)-.gamma.-amino-.alpha.-hydroxybutyryl]kanamycin A or a
nontoxic pharmaceutically acceptable acid addition salt thereof,
which comprises acylating polysilylated kanamycin A containing
carbobenzyloxy groups on the 3-amino and 6'-amino moieties with a
mixed acid anhydride of
L-(-)-.gamma.-benzyloxycarbonylamino-.alpha.-hydroxybutyric acid
(and preferably its mixed acid anhydride with pivalic acid, benzoic
acid, isobutylcarbonic acid or benzylcarbonic acid) in a
substantially anhydrous organic solvent, and subsequently removing
all blocking groups.
In another most preferred embodiment, this invention relates to the
preparation of
1-N-[L-(-)-.gamma.-amino-.alpha.-hydroxybutyryl]kanamycin A or a
nontoxic pharmaceutically acceptable acid addition salt thereof,
which comprises acylating polysilylated kanamycin A with an active
ester of
L-(-)-.gamma.-benzyloxycarbonylamino-.alpha.-hydroxybutyric acid
(and preferably its active ester with N-hydroxysuccinimide,
N-hydroxy-5-norbornene-2,3-dicarboximide or N-hydroxyphthalimide)
in a substantially anhydrous organic solvent, and subsequently
removing all blocking groups.
In another most preferred embodiment, this invention relates to the
preparation of
1-N-[L-(-)-.gamma.-amino-.alpha.-hydroxybutyryl]kanamycin A or a
nontoxic pharmaceutically acceptable acid addition salt thereof,
which comprises acylating polysilylated kanamycin A containing a
carbobenzyloxy group on the 6'-amino moiety with an active ester of
L-(-)-.gamma.-benzyloxycarbonylamino-.alpha.-hydroxybutyric acid
(and preferably its active ester with N-hydroxysuccinimide,
N-hydroxy-5-norbornene-2,3-dicarboximide or N-hydroxyphthalimide)
in a substantially anhydrous organic solvent, and subsequently
removing all blocking groups.
In another most preferred embodiment, this invention relates to the
preparation of
1-N-[L-(-)-.gamma.-amino-.alpha.-hydroxybutyryl]kanamycin A or a
nontoxic pharmaceutically acceptable acid addition salt thereof,
which comprises acylating polysilylated kanamycin A containing
carbobenzyloxy groups on the 3-amino and 6'-amino moieties with an
active ester of
L-(-)-.gamma.-benzyloxycarbonylamino-.alpha.-hydroxybutyric acid
(and preferably its active ester with N-hydroxysuccinimide,
N-hydroxy-5-norbornene-2,3-dicarboximide or N-hydroxyphthalimide)
in a substantially anhydrous organic solvent, and subsequently
removing all blocking groups.
In another aspect, the present invention provides polysilylated
kanamycin A or B or polysilylated kanamycin A or B containing a
conventional non-silyl blocking group on the 3-amino group, the
6'-amino group or the 3-amino and 6'-amino groups. In a preferred
embodiment the material is polysilylated kanamycin A or B (and
preferably polysilylated kanamycin A) containing an average number
of silyl groups (and preferably trimethylsilyl groups) per molecule
of from 4 to 8. In another preferred embodiment the material is
polysilylated kanamycin A or B (and preferably polysilylated
kanamycin A) containing a conventional non-silyl blocking group on
the 6'-amino group and containing an average number of silyl groups
(and preferably trimethylsilyl groups) per molecule of from 3 to 7.
In another preferred embodiment the material is polysilylated
kanamycin A or B (and preferably polysilylated kanamycin A)
containing conventional non-silyl blocking groups on the 3-amino
and 6'-amino groups and containing an average number of silyl
groups (and preferably trimethylsilyl groups) per molecule of from
3 to 6.
When the material is polysilylated kanamycin A or B containing a
non-silyl blocking group on the 3- and/or 6'-amino moiety, said
blocking group is preferably selected from those of the formulae
##STR8## wherein R.sup.1 and R.sup.2 are alike or different and
each is H, F, Cl, Br, NO.sub.2, OH (lower)alkyl or (lower)alkoxy, X
is Cl, Br, F or I, and Y is H, Cl, Br, F or I. The most preferred
blocking group is the carbobenzyloxy group.
As used herein and in the claims, the term "nontoxic,
pharmaceutically acceptable acid addition salt" of a compound of
Formula I means a mono-, di-, tri- or tetrasalt formed by the
interaction of one molecule of a compound of Formula I with 1-4
equivalents of a nontoxic, pharmaceutically acceptable acid.
Included among these acids are acetic, hydrochloric, sulfuric,
maleic, phosphoric, nitric, hydrobromic, ascorbic, malic and citric
acid, and those other acids commonly used to make salts of
amine-containing pharmaceuticals.
Acylation of the polysilylated kanamycin A or B starting material
(with or without a non-silyl blocking group on the 3- and/or
6'-amino moiety) may, in general, be conducted in an organic
solvent in which the starting material has sufficient solubility.
These starting materials are highly soluble in most common organic
solvents. Suitable solvents include for example, acetone, diethyl
ketone, methyl n-propyl ketone, methyl isobutyl ketone, methyl
ethyl ketone, acetonitrile, heptane, glyme, diglyme, dioxane,
toluene, tetrahydrofuran, cyclohexanone, pyridine, methylene
chloride, chloroform, carbon tetrachloride and mixtures of
acetone/butanol or diethyl ketone/butanol. The choice of solvent is
dependent on the particular starting materials employed. Ketones,
generally, are the preferred solvents. The most advantageous
solvent for the particular combination of reactants being utilized
can readily be determined by routine experimentation.
Suitable silylating agents for use in preparing the polysilylated
kanamycin starting materials utilized herein include those of the
formula ##STR9## wherein R.sup.5, R.sup.6 and R.sup.7 are selected
from the group consisting of hydrogen, halogen, (lower)alkyl,
(lower)alkoxy, halo(lower)alkyl and phenyl, at least one of the
said R.sup.5, R.sup.6 and R.sup.7 groups being other than halogen
or hydrogen; R.sup.4 is (lower)alkyl, m is an integer of 1 to 2 and
X is selected from the group consisting of halogen and ##STR10##
wherein R.sup.8 is hydrogen or (lower)alkyl and R.sup.9 is
hydrogen, (lower)alkyl or ##STR11## in which R.sup.5, R.sup.6 and
R.sup.7 are as defined above.
Specific silyl compounds of Formulas IV and V are:
trimethylchlorosilane, hexamethyldisilazane, triethylchlorosilane,
methyltrichlorosilane, dimethyldichlorosilane, triethylbromosilane,
tri-n-propylchlorosilane, methyldiethylchlorosilane,
dimethylethylchlorosilane, dimethyl-t-butylchlorosilane,
phenyldimethylbromosilane, benzylmethylethylchlorosilane,
phenylethylmethylchlorosilane, triphenylchlorosilane,
triphenylfluorosilane, tri-o-tolylchlorosilane,
tri-p-dimethylaminophenylchlorosilane, N-ethyltriethylsilylamine,
hexaethyldisilazane, triphenylsilylamine, tri-n-propylsilylamine,
tetraethyldimethyldisilazane, hexaphenyldisilazane,
hexa-p-tolyldisilazane, etc. Also useful are
hexaalkylcyclotrisilazanes and octa-alkylcyclotetrasilazanes. Other
suitable silylating agents are silylamides (such as
trialkylsilylacetamides and bis-trialkylsilylacetamides),
silylureas (such as trimethylsilylurea) and silylureides.
Trimethylsilylimidazole also may be utilized.
A preferred silyl group is the trimethylsilyl group and preferred
silylating agents for introducing the trimethylsilyl group are
hexamethyldisilazane, bis(trimethylsilyl)acetamide,
trimethylsilylacetamide and trimethylchlorosilane.
Hexamethyldisilazane is most preferred.
When utilizing polysilylated kanamycin A or B containing a
non-silyl blocking group on the 3- and/or 6'-amino moiety as a
starting material, said starting material may be prepared either by
polysilylating the desired N-blocked kanamycin A or B, or by
introducing the desired N-blocking group into polysilylated
kanamycin A or B.
Methods for the introduction of silyl groups into organic
compounds, including certain aminoglycosides, are known in the art.
The polysilylated kanamycins (with or without a non-silyl blocking
group on the 3- and/or 6'-amino moiety) may be prepared by methods
which are known per se, or as described in this specification.
As used herein, the term polysilylated kanamycin A or B refers to
kanamycin A or B containing from two to ten silyl groups in the
molecule. Thus, the term polysilylated kanamycin A or B does not
include persilylated kanamycin A or B, which would contain eleven
silyl groups in the molecule. Similarly, polysilylated kanamycin A
or B containing a single non-silyl blocking group on the 3-amino or
6'-amino moiety includes the N-blocked kanamycin containing from
two to nine silyl groups (and excludes the persilylated compound
which would contain ten silyl groups), while polysilylated
kanamycin A or B containing non-silyl blocking groups on both the
3-amino and 6'-amino moieties includes the di-N-blocked kanamycin
containing from two to eight silyl groups (and excludes the
persilylated compound which would contain nine silyl groups).
The precise number of silyl groups (or their location) present in
the polysilylated kanamycin starting materials (with or without a
non-silyl blocking group on the 3- and/or 6'-amino moiety) is not
known. We have found that both undersilylation and oversilylation
lower the yield of the desired product and increase the yield of
other products. In the case of gross under- or oversilylation,
little or none of the desired product may be formed. The degree of
silylation which will give the greatest yield of desired product
will depend on the particular reactants being used in the acylation
step. The most advantageous degree of silylation using any
combination of reactants can readily be determined by routine
experimentation.
When preparing
1-N-[L-(-)-.gamma.-amino-.alpha.-hydroxybutyryl]kanamycin A by
acylating polysilylated kanamycin A with the N-hydroxysuccinimide
ester of
L-(-)-.gamma.-benzyloxycarbonylamino-.alpha.-hydroxybutyric acid in
acetone solution, we have found that good yields of the desired
product are obtained by utilizing polysilylated kanamycin A which
has been prepared by reacting from about 4 to about 5.5 moles of
hexamethyldisilazane per mole of kanamycin A. Greater or lesser
amounts of hexamethyldisilazane may be utilized, but the yield of
desired product in the subsequent acylation step is lowered
significantly. In the specific process set forth above we prefer to
utilize from about 4.5 to about 5.0 moles of hexamethyldisilazane
per mole of kanamycin in order to obtain maximum yield of product
in the acylation step.
It will be appreciated that each mole of hexamethyldisilazane is
capable of introducing two equivalents of the trimethylsilyl group
into kanamycin A or B. Kanamycin A and B each have a total of
eleven sites (NH.sub.2 and OH groups) which might be silylated,
while kanamycin A and B containing a non-silyl blocking group on
the 3-amino or 6'-amino moiety each have a total of 10 such sites,
and kanamycin A and B containing non-silyl blocking groups on both
the 3-amino and 6'-amino moieties each contain 9 such sites. Thus,
5.5 moles of hexamethyldisilazane per mole of kanamycin A or B
could theoretically completely silylate all OH and NH.sub.2
moieties of the kanamycin, while 5.0 moles of hexamethyldisilazane
could completely silylate one mole of kanamycin A or B containing a
single non-silyl blocking group, and 4.5 moles of
hexamethyldisilazane could completely silylate one mole of
kanamycin A or B containing two non-silyl blocking groups. However,
we believe that such extensive silylation does not take place with
these molar ratios during reasonable reaction time periods,
although higher degrees of silylation are obtained in a given
reaction time when a silylation catalyst is added.
Silylation catalysts greatly accelerate the rate of silylation.
Suitable silylation catalysts are well known in the art and include
inter alia amine sulfates (e.g. kanamycin sulfate), sulfamic acid,
imidazole and trimethylchlorosilane. Silylation catalysts generally
promote a higher degree of silylation than is required in the
process of this invention. However, oversilylated kanamycin A or B
can be used as starting material if it is first treated with a
desilylating agent to reduce the degree of silylation before the
acylation reaction is carried out.
Good yields of desired product are obtained when acylating
polysilylated kanamycin A prepared using a 5.5:1 molar ratio of
hexamethyldisilazane to kanamycin A. However, when kanamycin A
silylated with a 7:1 molar ratio of hexamethyldisilazane (or with a
5.5:1 molar ratio in the presence of a silylation catalyst) was
acylated in acetone with the N-hydroxysuccinimide ester of
L-(-)-.gamma.-benzyloxycarbonylamino-.alpha.-hydroxybutyric acid,
less than a 1% yield of the desired product was obtained. However,
when this same "oversilylated" kanamycin A was acylated with the
same acylating agent in acetone solution to which water [21 moles
water per mole of kanamycin; 2.5% water (W/V)] had been added as a
desilylating agent 1 hour before acylation, a yield of
approximately 40% of the desired product was obtained. The same
results are obtained if the water is replaced by methanol or other
active hydrogen compound capable of effecting desilylation, e.g.
ethanol, propanol, butanediol, methyl mercaptan, ethyl mercaptan,
phenyl mercaptan, or the like.
Although it is usual to utilize dry solvents when working with
silylated materials, we have surprisingly found that, even in the
absence of "oversilylation", the addition of water to the reaction
solvent prior to acylation often gives equally good yields, and
sometimes gives better yields of desired product than in a dry
solvent. In acylation reactions conducted in acetone at the usual
concentrations of 10-20% (W/V) of polysilylated kanamycin A, we
have found that excellent yields of
1-N-[L-(-)-.gamma.-amino-.alpha.-hydroxybutyryl]kanamycin A were
obtained when adding up to 28 moles of water per mole of
polysilylated kanamycin A; at 20% concentration, 28 moles per mole
is approximately 8% water. With other combinations of reactants and
solvents, even more water may be tolerated or be beneficial. The
acylation reaction may be conducted in solvents containing up to
about 40% water, although at such high water concentrations one
must utilize short acylation times in order to avoid excessive
desilylation of the polysilylated kanamycin A or B starting
material. Accordingly, as used herein and in the claims, the term
"substantially anhydrous organic solvent" is intended to include
solvents containing up to about 40% water. A preferred range is up
to about 20% water, a more preferred range is up to about 8% water
and the most preferred range is up to about 4% water.
As indicated above, the most desirable degree of silylation for any
combination of acylation reactants may be readily determined by
routine experimentation. It is believed that the preferred average
number of silyl groups in the starting material will usually be
between 4 and 8 for kanamycin A or B, between 3 and 7 for kanamycin
A or B containing a single non-silyl blocking group and between 3
and 6 for kanamycin A or B containing two non-silyl blocking
groups, but this is only theory and is not considered an essential
part of this invention.
Except as described above for solvents containing very high water
levels, the duration of the acylation reaction is not critical.
Temperatures in the range of about -30.degree. C. to about
100.degree. C. may be used for reaction times ranging from about
one hour up to a day or more. The reaction usually proceeds well at
room temperature and, for convenience, may be conducted at ambient
temperature. However, for maximum yields and selective acylation,
we prefer to conduct the acylation at from about 0.degree. to
5.degree..
Acylation of the 1-amino moiety of the polysilylated kanamycin A or
B (with or without a non-silyl blocking group on the 3- and/or
6'-amino moiety) may be conducted with any acylating derivative of
the acid of Formula II which is known in the art to be suitable for
the acylation of a primary amino group. Examples of suitable
acylating derivatives of the free acid include the corresponding
acid anhydrides, mixed anhydrides, e.g. alkoxyformic anhydrides,
acid halides, acid azides, active esters and active thioesters. The
free acid may be coupled with the polysilylated kanamycin starting
material after first reacting said free acid with
N,N'-dimethylchloroformininium chloride [cf. Great Britain No.
1,008,170 and Novak and Weichet, Experientia XXI, 6, 360 (1965)] or
by the use of an N,N'-carboxyldiimidazole or an
N,N'-carbonylditriazole [cf. South African Specification No.
63/2684] or a carbodiimide reagent [especially
N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide or
N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide: cf. Sheehan and
Hess, J.A.C.S., 77, 1967 (1955)], or of an alkynylamine reagent
[cf. R. Buijle and H. G. Viehe, Angew. Chem. International Edition,
3, 582, (1964)] or of an isoxazolium salt reagent [cf. R. B.
Woodward, R. A. Olofson and H. Mayer, J. Amer. Chem. Soc., 83, 1010
(1961)], or of a ketenimine reagent [cf. C. L. Stevens and M. E.
Munk, J. Amer. Chem. Soc., 80, 4065 (1958)] or of
hexachlorocyclotriphosphatriazine or
hexabromocyclotriphosphatriazine (U.S. Pat. No. 3,651,050) or of
diphenylphosphoryl azide [DDPA; J. Amer. Chem. Soc., 94, 6203-6205
(1972)] or of diethylphosphoryl cyanide [DEPC; Tetrahedron Letters
No. 18, pp. 1595-1598 (1973)] or of diphenyl phosphite [Tetrahedron
Letters No. 49, pp. 5047-5050 (1972)]. Another equivalent of the
acid is a corresponding azolide, i.e., an amide of the
corresponding acid whose amide nitrogen is a member of a
quasiaromatic five membered ring containing at least two nitrogen
atoms, i.e., imidazole, pyrazole, the triazoles, benzimidazole,
benzotriazole and their substituted derivatives. As will be
appreciated by those skilled in the art, it sometimes may be
desirable or necessary to protect the hydroxyl group of the
acylating derivative of the acid of Formula II, e.g. when utilizing
acylating derivatives such as an acid halide. Protection of the
hydroxyl group may be accomplished by means known in the art, e.g.
by use of a carbobenzyloxy group, by acetylation, by silylation, or
the like.
After completion of the acylation reaction, all blocking groups are
removed by methods known per se to yield the desired product of
Formula I. The silyl groups may, for example, readily be removed by
hydrolysis with water, preferably at low pH. Blocking group B of
the acylating derivatives of the acid of Formula II, and the
blocking group on the 3- and/or 6'-amino moiety of the
polysilylated kanamycin starting material (if present) may also be
removed by known methods. Thus, a t-butoxycarbonyl group may be
removed by the use of formic acid, a carbobenzyloxy group by
catalytic hydrogenation, a 2-hydroxy-1-naphthcarbonyl group by acid
hydrolysis, a trichloroethoxycarbonyl group by treatment with zinc
dust in glacial acetic acid, the phthaloyl group by treatment with
hydrazine hydrate in ethanol under heating, the trifluoroacetyl
group by treatment with NH.sub.4 OH, etc.
Yields of product were determined by various methods. After removal
of all blocking groups and chromatography on a CG-50 (NH.sub.4 +)
column, the yield of BB-K8 could be determined by isolation of the
crystalline solid from the appropriate fractions or by
microbiological assay (turbidimetric or plate) of the appropriate
fractions. Another technique which we utilized was high performance
liquid chromatography of the unreduced acylation mixture, i.e. the
aqueous solution obtained after hydrolysis of the silyl groups and
removal of organic solvent but before hydrogenolysis to remove the
remaining blocking group(s). This assay was not a direct assay for
BB-K8 or BB-K29, but for the corresponding mono- or di-N-blocked
compounds.
The instrument utilized was a Waters Associates ALC/GPC 244 high
pressure liquid chromatograph with a Waters Associates Model 440
absorbance detector and a 30 cm.times.3.9 mm i.d. .mu.-Bondapak
C-18 column, under the following conditions:
______________________________________ Mobile Phase 25% 2-propanol
75% 0.01M sodium acetate pH 4.0 Flow Rate 1 ml./minute Detector UV
at 254 nm. Sensitivity 0.04 AUFS Diluent DMSO Injected Amount 5
.mu.l Concentration 10 mg./ml.
______________________________________
Chart speed varied, but 2 minutes/inch was typical. The above
conditions gave UV traces with peaks which were easy to measure
quantitatively. The results of the above analyses are referred to
in the specification as HPLC assays.
In order to avoid the repetition of complex chemical names, the
following abbreviations are sometimes utilized in this
specification.
______________________________________ AHBA
L-(-)-.gamma.-amino-.alpha.-hydroxybutyric acid BHBA
N--Carbobenzyloxy derivative of AHBA HONB
N--hydroxy-5-norbornene-2,3-dicarboximide NAE
N--hydroxy-5-norbornene-2,3-dicarboximide (or BHBA--`ONB`)
activated ester of BHBA HONS N--hydroxysuccinimide SAE
N--hydroxysuccinimide activated ester of (or BHBA--`ONS`) BHBA DCC
dicyclohexylcarbodiimide DCU dicyclohexylurea HMDS
hexamethyldisilazane BSA bis(trimethylsilyl)acetamide MSA
trimethylsilylacetamide TFA trifluoroacetyl t-BOC tert.
butyloxycarbonyl ______________________________________
"Dicalite" is a trademark of the Great Lakes Carbon Corporation for
diatomaceous earth.
"Amberlite CG-50" is a Trademark of the Rohm & Haas Co. for the
chromatographic grade of a weakly acid cationic exchange resin of
the carboxylic-polymethacrylic type.
".mu.-Bondapak" is a Trademark of Waters Associates for a series of
high performance liquid chromatography columns.
All temperatures herein are given in degrees centigrade. As used
herein, the terms "(lower)alkyl" and "(lower)alkoxy" refer to alkyl
or alkoxy groups containing from 1 to six carbon atoms.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
EXAMPLE 1
Preparation of
1-N-[L-(-)-.gamma.-Amino-.alpha.-hydroxybutyryl]kanamycin A (BB-K8)
by Selective Acylation of Poly(trimethylsilyl)
6'-N-Carbobenzyloxykanamycin A in Anhydrous Diethyl Ketone
6'-N-Carbobenzyloxykanamycin A (15 g., 24.24 m. moles) was slurried
in 90 ml. of dry acetonitrile and heated to reflux under a nitrogen
atmosphere. Hexamethyldisilazane (17.5 g., 108.48 m. moles) was
added slowly over 30 minutes, and the resulting solution was
refluxed for 24 hours. After removal of the solvent in vacuo
(40.degree.) and complete drying under vacuum (10 mm), 27.9 g. of a
white, amorphous solid was obtained [90.71% calculated as
6'-N-Carbobenzyloxykanamycin A (Silyl).sub.9 ].
This solid was dissolved in 150 ml. of dry diethyl ketone at
23.degree..
L-(-)-.gamma.-benzyloxycarbonylamino-.alpha.-hydroxybutyric acid
N-hydroxy-5-norbornene-2,3-dicarboximide ester (NAE) (11.05 g.,
26.67 m. moles) dissolved in 100 ml. of dry diethyl ketone at
23.degree. was added slowly with good agitation over 1/2 hour. The
solution was stirred at 23.degree. for 78 hours. The yellow, clear
solution (pH 7.0) was diluted with 100 ml. of water. The pH of the
mixture was adjusted to 2.8 (3 N HCl) and stirred vigorously at
23.degree. for 15 minutes. The aqueous phase was separated, and the
organic phase was extracted with 50 ml. of pH 2.8 water. The
combined aqueous fractions were washed with 50 ml. of ethyl
acetate. The solution was placed in a 500 ml. Parr bottle, together
with 5 g. of 5% palladium on carbon catalyst (Engelhard) and
reduced at 50 psi H.sub.2 for 2 hours at 23.degree.. The mixture
was filtered through a pad of Dicalite which was then washed with
an additional 30 ml. of water. The colorless filtrate was
concentrated in vacuo (40.degree.-45.degree.) to 50 ml. The
solution was charged on a 5.times.100 cm CG-50 (NH.sub.4 +) ion
exchange column. After washing with 1000 ml. of water, unreacted
kanamycin A,
3-[L-(-)-.gamma.-amino-.alpha.-hydroxybutyryl]kanamycin A (BB-K29)
and BB-K8 were eluted with 0.5 N ammonium hydroxide. Polyacyl
material was recovered with 3 N ammonium hydroxide. Bioassay, thin
layer chromatography and optical rotation were used to monitor the
progress of elution. The volume and observed optical rotation of
each fraction of eluate, as well as the weight and percent yield of
solid isolated from each fraction by evaporation to dryness, are
summarized below:
______________________________________ Volume Weight Material (ml)
.alpha..sub.578 (gms.) % Yield
______________________________________ Kanamycin A 1000 +0.115
0.989 9.15 BB-K29 1750 +0.24 4.37 32.0 BB-K8 2000 +0.31 6.20 47.4
Polyacyls 900 +0.032 0.288 2.0
______________________________________
The spent diethyl ketone layer was shown by high performance liquid
chromatography to contain an additional 3-5% BB-K8.
The crude BB-K8 (6.20 gms.) was dissolved in 20 ml. of water and
diluted with 20 ml of methanol, and 20 ml. of isopropanol was added
to induce crystallization. There was obtained 6.0 gms. (45.8%) of
crystalline BB-K8.
EXAMPLE 2
Preparation of
1-N-[L-(-)-.gamma.-Amino-.alpha.-hydroxybutyryl]kanamycin A (BB-K8)
by Selective Acylation of Poly(trimethylsilyl)
6'-N-Carbobenzyloxykanamycin A in Anhydrous Acetone
Poly(trimethylsilyl) 6'-N-carbobenzyloxy kana A prepared as in
Example 1 (103 g., 0.081 moles, calculated as
6'-N-Carbobenzyloxykanamycin A (Silyl).sub.9) was dissolved in 100
ml. of dry acetone at 23.degree..
L-(-)-.gamma.-benzyloxycarbonylamino-.alpha.-hydroxybutyric acid
N-hydroxy-5-norbornene-2,3-dicarboximide ester (NAE) (35.24 g.,
0.085 moles) dissolved in 180 ml. of dry acetone at 23.degree. was
added slowly with good agitation to the solution of
poly(trimethylsilyl) 6'-N-Carbobenzyloxykanamycin A over a 15
minute period. The solution was stirred at 23.degree. for 20 hours
under a nitrogen atmosphere. The pale yellow, clear solution (pH
7.2) was diluted with 100 ml. of water. The pH of the mixture was
adjusted to 2.5 (3 N HCl) and stirring continued at 23.degree. for
15 minutes. Acetone was removed using steam-ejector vacuum at about
35.degree.. The solution was placed in a 500 ml. Parr bottle,
together with 10 g. of 5% palladium on carbon catalyst (Engelhard)
and reduced at 40 psi H.sub.2 for 2 hours at 23.degree.. The
mixture was filtered through a pad of diatomaceous earth which was
then washed with an additional 50 ml. of water. After concentration
to approximately 1/3 volume, the solution (pH 6.0-7.2) was charged
on a 6.times.110 cm. CG-50 (NH.sub.4 +) ion exchange column and
eluted with a stepwise gradient from H.sub.2 O to 0.6 N ammonium
hydroxide to recover BB-K8. An automatic polarimeter was used to
monitor the progress of elution. Combinations were made on the
basis of thin layer chromatography evaluation. the combined BB-K8
fractions were concentrated to 25-30% solids. The solution was
diluted with an equal volume of methanol, followed by two volumes
of isopropanol to induce crystallization. There was recovered 18.2
g. (40%) of crystalline BB-K8.
The recovery of 12% kanamycin A, 40% BB-K29 and 5% polyacylated
kanamycin gave a material balance of 97%.
EXAMPLE 3
Preparation of
1-N-[L-(-)-.gamma.-Amino-.alpha.-hydroxybutyryl]kanamycin A (BB-K8)
by Selective Acylation of Poly(trimethylsilyl) Kanamycin A, Using
In Situ Blocking
A. Poly(trimethylsilyl) Kanamycin A
Kanamycin A free base (18 g. activity, 37.15 m. moles) was slurried
in 200 ml. of dry acetonitrile and heated to reflux.
Hexamethyldisilazane (29.8 g., 184.6 m. moles) was added over 30
minutes and the mixture was stirred at reflux for 78 hours to give
a light yellow clear solution. Removal of the solvent under vacuum
left an amorphous solid residue (43 gm., 94%) [calculated as
kanamycin A (silyl).sub.10 ].
B. 1-N-[L-(-)-.gamma.-Amino-.alpha.-hydroxybutyryl]kanamycin A
p-(Benzyloxycarbonyloxy)benzoic acid (5.56 g., 20.43 m. moles) was
slurried in 50 ml. of dry acetonitrile at 23.degree..
N,O-bis-Trimethylsilyl acetamide (8.4 g., 41.37 m. mole) was added
with good stirring. The solution was held for 30 minutes at
23.degree., and then added over 3 hours with vigorous stirring to a
solution of poly(trimethylsilyl)kanamycin A (21.5 g., 17.83 m.
mole, calculated as the (silyl).sub.10 compound) in 75 ml. of dry
acetonitrile at 23.degree.. The mix was stirred for 4 hours, the
solvent was removed in vacuo (40.degree.), and the oily residue was
dissolved in 50 ml. of dry acetone at 23.degree. C.
L-(-)-.gamma.-benzyloxycarbonylamino-.alpha.-hydroxybutyric acid
N-hydroxy-5-norbornene-2,3-dicarboximide ester (NAE) (8.55 g.,
20.63 m. moles) in 30 ml. of acetone was added to the above
solution over a period of 5 minutes. The mixture was held at
23.degree. C. for 78 hours. The solution was diluted with 100 ml.
of water and the pH (7.0) lowered to 2.5 (6 N HCl). The mixture was
placed in a 500 ml. Parr bottle together with 3 g. of 5% palladium
on carbon catalyst (Engelhard) and reduced at 40 psi H.sub.2 for 2
hours at 23.degree.. The mixture was filtered through a pad of
diatomaceous earth which was then washed with 20 ml. of water. The
combined filtrate and washings (168 ml.) were determined by
microbiological assay against E. coli to contain approximately
11,400 mcg/ml. (19% yield) of BB-K8.
EXAMPLE 4
Preparation of 1-N-[L-(-)-.gamma.-Amino-.alpha.-hydroxybutyryl]
kanamycin A (BB-K8) by Selective Acylation of
Poly(trimethylsilyl)Kanamycin A
A. Poly(trimethylsilyl) Kanamycin A
A suspension of 10 g. (20.6 m. moles) kanamycin A in 100 ml. of dry
acetonitrile and 25 ml. (119 m. moles)
1,1,1,3,3,3-hexamethyldisilazane was refluxed for 72 hours. A clear
light yellow solution resulted. The solution was stripped to
dryness in vacuo at 30.degree.-40.degree. C. There was obtained
21.3 g. of poly(trimethylsilyl) Kanamycin A as a light tan
amorphous powder [85% yield calculated as kanamycin A
(silyl).sub.10 ].
B. 1-N-[L-(-)-.gamma.-Amino-.alpha.-hydroxybutyryl]kanamycin A
To a solution of 2.4 g. (2.0 m. moles) of poly(trimethylsilyl)
Kanamycin A in 30 ml. of dry acetone was added slowly 2.0 m. moles
of L-(-)-.gamma.-benzyloxycarbonylamino-.alpha.-hydroxybutyric acid
N-hydroxy-5-norbornene-2,3-dicarboximide ester (NAE) in 10 ml. of
dry acetone at 0.degree.-5.degree. C. The reaction mixture was
stirred at 23.degree. C. for a week and then stripped to dryness in
vacuo at a bath temperature of 30.degree.-40.degree. C. Water (60
ml.) was then added to the residue, followed by 70 ml. of methanol
to obtain a solution. The solution was acidified with 3 N HCl to pH
2.0 and then reduced at 50 psi H.sub.2 for 2 hours, using 500 mg of
5% palladium on carbon catalyst. The material was filtered, and the
combined filtrate and washings were determined by microbiological
assay against E. coli to contain a 29.4% yield of BB-K8.
EXAMPLE 5
Preparation of BB-K8 by Selective N-Acylation of Polytrimethylsilyl
6'-N-Carbobenzoxy Kanamycin A in Anhydrous Acetone
I. Summary
Silylation of 6'-N-carbobenzoxy Kana A in acetonitrile using
hexamethyldisilazane (HMDS) affords the 6'-N-carbobenzoxy Kana A
(silyl).sub.9 intermediate .circle. . This silylated Kana A is
readily soluble in most organic solvents. Acylation with NAE in
anhydrous acetone at 23.degree. using a 5% molar excess of NAE
relative to 6'-N-Cbz Kana A input afforded a mixture containing
only Cbz derivatives of BB-K8 and BB-K29, some unreacted Kana A and
some polyacyl material. No BB-K11 was detectable in any of these
studies. Elution of an acetone acylation mix, after reduction and
workup, from a CG-50 (NH.sub.4 +) column using an ammonium
hydroxide gradient afforded isolated yields of pure BB-K8 in the
40% range.
______________________________________ II. Equations ##STR12## A.
6'-NCbz Kana A C.sub.26 H.sub.42 O.sub.13 N.sub.4 (618.65) +
(CH.sub.3).sub.3 SiNHSi(CH.sub.3).sub.3 HMDS (161.4) ##STR13##
##STR14## R = Si(CH.sub.3).sub.3 .circle.1 6'-NCbz Kana A
(Silyl).sub.9 C.sub.53 H.sub.114 O.sub.13 N.sub.4 Si.sub.9 (1268.3)
##STR15## B. ##STR16## ##STR17## .circle.2 NAE (414.6) ##STR18## C.
.THorizBrace. ##STR19## ##STR20## BB-K8
______________________________________ III. Materials Wgt. g. Vol.
ml. Moles ______________________________________ 6'-NCbz Kana A 50
.081 HMDS 58.9 76.5 .365 Acetonitrile 300 BHBA 21.5 .085 HONB 15.2
.085 DCC 17.48 .085 Acetone 260 CG-50(NH.sub.4.sup.+) 3000 Methanol
As required IPA As required ______________________________________
IV. Safety 6'-NCbz Kana A No direct information avail- able. Avoid
dust contact. Acetonitrile Treat as a cyanide. Avoid breathing
vapors. May cause skin irritation. Hexamethyldisilazane Irritant,
handle with care. (HMDS) 6'-NCbz Kana A (Silyl).sub.9 No direct
information avail- able, handle with care. BHBA Toxicity is not
established. Avoid exposure to solids. HONB Toxicity unknown. Use
pre- caution in handling. DCC A severe skin and eye irritant. Avoid
inhalation of mist or vapors. Toxic. Acetone Flammable. Inhalation
may produce headache, fatigue, excitement, bronchial irritation,
and, in large amounts narcosis. NAE No direct information avail-
able; always handled directly as solution in acetone. Methanol
Flammable. Poisoning may occur from ingestion, inhal- ation or
percutaneous absorp- tion. Isopropanol Flammable. Ingestion or
inhalation of large quantities of the vapor may cause headache,
dizziness, mental depression, vomiting, narcosis. Ammonium
hydroxide Toxic vapors. Wear mask, avoid contact with liquid.
CG-50(NH.sub.4.sup. +) No toxicity data available, handle with
care. ______________________________________
V. Procedure
A. Preparation 6'-N-Carbobenzyloxykanamycin A (silyl).sub.9
[6'-N-Cbz Kana A (Silyl).sub.9 ]
1. Slurry 50 g. of 6'-N-carbobenzyloxykanamycin A (KF<4%) in 300
ml. of acetonitrile (KF<0.01%). Bring to reflux (74.degree.)
maintaining a stream of dry nitrogen through the slurry.
2. Add slowly over a 30 minute period 75.8 ml. hexamethyldisilazane
(HMDS). Complete solution will occur with evolution of ammonia
gas.
3. Continue refluxing for 18-20 hours under a nitrogen purge.
4. Concentrate the clear, light yellow solution under vacuum (bath
temp. 40.degree.-50.degree.) to a foamy solid. Yields of the
silyl.sub.9 compound 89-92 g. (90-94% Theory).
NOTE: For future reference; in other solvent studies this solid is
normally not isolated but used directly for the acylation.
B. Preparation of N-hydroxy-5-norbornene-2,3-dicarboximide ester of
L-(-)-.alpha.-carbobenzyloxyamino-.alpha.-hydroxybutyric acid
(NAE)
1. Dissolve 21.5 g. of
L-(-)-.gamma.-carbobenzyloxyamino-.alpha.-hydroxybutyric acid
(BHBA) in 100 ml. of dry acetone at 23.degree. followed by 15.2 g.
of N-hydroxy-5-norbornene-2,3-dicarboximide (HONB). A complete
solution will result.
2. Over 30 minutes add a solution of 17.48 g. of
dicyclohexylcarbodiimide (DCC) in 50 ml. of acetone with agitation.
The temperature will rise to approximately 40.degree. during the
addition with precipitation of dicyclohexylurea (DCU).
3. Agitate the slurry for 3-4 hours allowing the temperature to
equilibrate to 23.degree.-25.degree..
4. Remove the urea derivative by filtration; wash the cake with 30
ml. acetone. Save the filtrate plus washings for the acylation step
below.
C. Acylation of C'-N-Cbz Kana A (Silyl).sub.9
1. Dissolve the 6'-N-Cbz Kana A (silyl).sub.9 isolated in Part A,
Step 4 in 100 ml of dry acetone at 23.degree.-24.degree..
2. With good agitation slowly add the NAE solution prepared in Part
B over a 15 minute period. The temperature will gradually rise to
approximately 40.degree.. Allow the solution to equilibrate to
23.degree. and continue stirring for 18-20 hours under a nitrogen
atmosphere.
3. Add 100 ml. of water and lower the pH (6.9-7.2) to 2.2-2.5 with
6 N hydrochloric acid. Agitate for 15 minutes at 23.degree.. (NOTE:
A second layer may form--this does not present a problem in the
workup).
4. Remove acetone under vacuum at a bath temperature of
30.degree.-35.degree.. Transfer the concentrate to a suitable
hydrogenation vessel (prepurged with nitrogen). Add 10 g. 5%
palladium on carbon catalyst, and hydrogenate at 40 psi for 2-3
hours.
5. Filter the mixture through a Dicalite pad, washing the
hydrogenation vessel and cake with an additional 50 ml. water.
6. Concentrate the filtrate plus wash to approximately 1/3 volume
(50 ml.) under vacuum at 40.degree.-45.degree..
7. Check the pH. It should be in the range 6.9-7.2. If not, adjust
with 1 N ammonium hydroxide. Charge the mixture on a CG-50
(NH.sub.4 +) column (6.times.110 cm).
8. Wash the column with 1000 ml. of deionized water. Then elute
with 0.5-0.6 N ammonium hydroxide using an automatic polarimeter to
monitor the progress of elution. The order of elution is as
follows:
Residual Kana A.fwdarw.BB-K29.fwdarw.BB-K8.
No BB-K11 was detected in any of our acylation workups. Polyacyl
material i.e. the 1,3-diAHBA analog of Kana A, is recovered by
washing the column with 3 N ammonium hydroxide.
9. Combine the BB-K8 fractions and concentrate to 25-30% solids.
Dilute with 1 volume of methanol, and seed with BB-K8 crystals.
10. Add slowly over 2 hours 2 volumes of isopropanol (IPA) with
good stirring, and crystallize at 23.degree. for 6-8 hours.
11. Filter the solid, wash with 50 ml. of 1:1:2 water/methanol/IPA
mixture, and finally with 25 ml. IPA.
12. Dry in a vacuum over at 40.degree. for 12-16 hours. Yield:
17.3-19.0 g. (38-42%) of BB-K8 having the following properties:
TLC
CHCl.sub.3 -methanol-NH.sub.4 OH-water (1:4:2:1), 5.times.20 cm.
silica gel plates from Quantum Industries--one zone as detected
with ninhydrin (RF.about. 0.4).
______________________________________ Specific Rotation
______________________________________ [.alpha.] 23.degree. H.sub.2
O 0.1M NH.sub.4 OH 0.1M H.sub.2 SO.sub.4 589 + 101.6 + 101.9 +
103.5 C = 1.0% ______________________________________
13. The recovery of BB-K29 in this system was also 39-42%, residual
Kana A 10-14% and 1,3-di AHBA-Kana A approximately 5% to give a
material balance >95%.
EXAMPLE 6
Preparation of BBK8 by Selective N-Acylation of Polytrimethylsilyl
Kana A in Anhydrous Acetone
I. Summary
Silylation of Kana A `base` acetonitrile using hexamethyldisilazane
(HMDS) yielded polytrimethylsilyl Kana A. The extent of silylation
is as yet uncertain, but for the time being is assumed to be Kana A
(Silyl).sub.10. Polysilylated Kana A is readily soluble in most
organic solvents. Acylation with SAE in anhydrous acetone at
23.degree. using a 1:1 molar ratio of SAE relative to Kana A input
afforded a mixture containing Cbz derivatives of BBK8 and BBK29,
usually in the ratio 2-3/1; BBK6 (approximately 5-8%), unreacted
Kana A (15-20%) and some polyacyl material (approximately 5-10%).
Again, as was seen in our previous work on the acylation of
polytrimethylsilyl 6'-N-Carbobenzoxy Kana A, no BBK11 was detected
in any of these experiments. Reduction and work-up of an acetone
acylation mix, followed by chromatography on a CG-50-(NH.sub.4 +)
column using 0.5 N ammonium hydroxide, afforded isolated
crystalline BBK8 in the 34-39% range.
______________________________________ II. Equations ##STR21## A.
Kana A `base` C.sub.18 H.sub.36 O.sub.11 N.sub.4 (484.51) +
(CH.sub.3).sub.3 SiNHSi(CH.sub.3).sub.3 HMDS (161.4) ##STR22##
##STR23## R = Si(CH.sub.3).sub.3 .circle.1 Kana A (Silyl).sub.10
C.sub.48 H.sub.116 O.sub.11 N.sub.4 Si.sub.10 (1206.35) ##STR24##
B. ##STR25## ##STR26## .circle.2 SAE (350.33) ##STR27## C.
.THorizBrace. ##STR28## ##STR29## BB-K8
______________________________________ III. Materials Wgt. g. Vol.,
ml. Moles ______________________________________ Kana A `base` 50
.103 HMDS (Sp. gr. 0.774) 86.68 112 .537 Acetonitrile 600 SAE 35.03
.10 Acetone 850 CG-50(NH.sub.4.sup..sym.) 3000 Methanol As required
IPA As required ______________________________________ IV. Safety
Kana A `base` Known drug - usual caution advised. Kana A
(Silyl).sub.10 No direct information avail- able, handle with care.
Other materials See Example 5
______________________________________
V. Procedure
A. Preparation of Kana A (Silyl).sub.10
1. Slurry 50 g. of Kana A `base` (KF 2.5-3.5%) in 500 ml. of
acetonitrile (KF< 0.01%). Bring to reflux (74.degree.)
maintaining a stream of dry nitrogen through the slurry.
2. Add slowly over a 30 minute period 112 ml. hexamethyldisilazane
(HMDS). Complete solution will occur within 4-5 hours with
evolution of ammonia gas.
3. Continue refluxing for 22-26 hours under a nitrogen purge.
4. Concentrate the clear faint yellow solution under vacuum
(40.degree.) to a syrupy residue. Flush with an additional 100 ml.
acetonitrile, and dry completely under high vacuum for 3-6 hours.
Yields of whitish amorphous solid are 109-115 g. (90-95% of theory,
calculated as Kana A (Silyl).sub.10).
B. Preparation of N-Hydroxysuccinimide ester of
L-(-)-.alpha.-carbobenzyloxyamino-.alpha.-hydroxybutyric acid
(SAE)
1. Dissolve 100 g of
L(-)-.alpha.-benzyloxycarbonylamino-.alpha.-hydroxybutyric acid
(BHBA) and 45.38 g of N-hydroxysuccinimide (N-HOS) in 1300 ml of
ethyl acetate (KF<0.05%) with stirring at 23.degree. C.
2. Dissolve 81.29 g of dicyclohexylcarbodiimide (DCC) in 400 ml. of
ethyl acetate (KF<0.05%) at 23.degree. C. With good agitation
add this solution over 30 minutes to step 1 solution. The
temperature will rise to .about.40.degree.-42.degree. C. with
concurrent precipitation of dicyclohexylurea (DCU). Agitate the
slurry 3-4 hours allowing the temperature to equilibrate to
23.degree. C.
3. Filter the DCU; wash the cake with 250 ml. of ethyl acetate
(KF<0.05%). Discard the DCU cake. Save the filtrate and
washes.
4. Concentrate the filtrate plus washes to .about.500 ml. (in vacuo
at 30.degree.-35.degree. C.). Some product will crystallize
out.
5. Transfer the concentrate to a suitable vessel and add with
vigorous agitation 100 ml. of heptane. If necessary, add seed
crystals of SAE. Crystallization will begin almost immediately.
Agitate the slurry for 30 minutes at 23.degree. C.
6. Add, over 30 minutes, 400 ml. of heptane and agitate the slurry
4-5 hours at 23.degree. C.
7. Filter and wash the cake with 200 ml. of 3:1 heptane/ethyl
acetate followed by 100 ml. of heptane.
8. Dry in a vacuum oven at 30.degree.-35.degree. C. for 18-20
hours.
Yield is 110.1-131.4 g (80-95%).
MP--119.degree.-120.degree. with softening at 114.degree.
(Corr.).
TLC--4 acetone:12 benzene:1 CH.sub.3 CO.sub.2 H--Detection 1%
aqueous KMO.sub.4.
Rf--0.7 for SAE; 0.2 BHBA on 2.times.10 cm prescored silica gel
plates from Analtech Inc.
C. Acylation of Kana A (Silyl).sub.10
1. Dissolve the Kana A (Silyl).sub.10 isolated in Part A, Step 4 in
500 ml. dry acetone at 23.degree. C.
2. With good agitation add rapidly the SAE prepared in Part B
(35.03 g) as a 10% solution in dry acetone over a 5-10 minute
period. The temperature will rise approximately 5.degree.. Allow
the solution to equilibrate to 23.degree., and continue stirring
for 18-20 hours.
3. The light orange, clear solution is diluted with 400 ml. of
water, and the pH (7.0-7.5) lowered to 2.2-2.5 with 3 N
hydrochloric acid. The clear solution is now agitated at 23.degree.
for 15-30 minutes.
4. Acetone is removed under vacuum at a bath temperature of
30.degree.-35.degree. (a small amount of material may separate at
this point, but presents no problem). Transfer the concentrate to a
suitable hydrogenation vessel. Add 10 g 5% palladium on carbon
catalyst, and hydrogenate at 50 psi for 2-3 hours.
5. Filter the mixture through a Dicalite pad, and wash the
hydrogenation vessel and cake with an additional 2.times.50 ml.
water.
6. Concentrate the filtrate plus washings to approximately 1/3
volume (150-165 ml.) under vacuum at 40.degree.-45.degree..
7. The pH at this point is in the range 6.0-7.0. The mixture is
charged on a CG-50(NH.sub.4 +) column (6.times.110 cm).
8. Wash the column with 1000 ml. of deionized water. Elute with 0.5
N ammonium hydroxide using an automatic polarimeter to monitor the
progress of elution. The order of elution is as follows:
Residual Kana A.fwdarw.BB-K6.fwdarw.BB-K29.fwdarw.BB-K8.
No BB-K11 was detected in any of our experiments.
9. Combine the BB-K8 fractions and concentrate to 25-30% solids.
Dilute with 1 volume methanol, and seed with BB-K8 crystals.
10. Add slowly over 2 hours 2 volumes of IPA with good stirring and
crystallize at 23.degree. for 6-8 hours.
11. Filter the solid, wash with 35 ml. of 1:1:2 water/methanol/IPA,
and finally with 35 ml. IPA.
12. Dry in a vacuum over at 40.degree. for 16-24 hours. Yield:
19.91-22.84 g (34-39%) IR, PMR and CMR spectral data in addition to
specific rotation were completely consistent for the desired
structure.
TLC System
CHCl.sub.3 /methanol/NH.sub.4 OH/water (1:4:2:1) 5.times.20 cm.
silica gel plates from Quantum Industries--1 Zone (BB-K8) having
R.sub.f .about.0.4 (Ninhydrin Detection).
EXAMPLE 7
Preparation of BB-K8 by Acylation of Poly(trimethylsilyl) 6'-N-Cbz
Kana A in Tetrahydrofuran With the Mixed Acid Anhydride of Pivalic
Acid and BHBA
A. Preparation of Mixed Anhydride
BHBA (5.066 gm., 20.0 m moles), BSA (4.068 gm., 20.0 m moles) and
triethylamine (2.116 g, 22.0 m moles) were dissolved in 200 ml. of
sieve dried tetrahydrofuran. The solution was refluxed for 21/4
hours and then chilled to -10.degree. C. Pivaloyl chloride (2.412
gm., 20.0 m moles) was added over a period of 2-3 minutes, with
stirring, and stirring was continued for 2 hours at -10.degree. C.
The temperature was then allowed to climb to 23.degree. C.
B. Acylation of Poly(trimethylsilyl) 6'-N-Cbz Kana A
Poly(trimethylsilyl) 6'-N-Cbz Kana A prepared as in Example 1
(5.454 gm., 4.97 m moles, calculated as 6'-Cbz Kana A
(silyl).sub.9) was dissolved in 50 ml. dry (molecular sieve)
tetrahydrofuran at 23.degree. C. One-half of the solution of mixed
anhydride prepared in step A, above, (10.0 m moles) was added over
a period of twenty minutes, with stirring, and stirring was
continued for 7 days.
Water (100 ml.) was then added to the reaction mixture, and the pH
(5.4) was adjusted to 2.0 with 3 M H.sub.2 SO.sub.4. Stirring was
continued for 1 hour and the solution was extracted with ethyl
acetate. Polyacylated material began to crystallize, so the
reaction mixture was filtered. After drying over P.sub.2 O.sub.5,
the recovered solids weighed 0.702 gms. The extraction of the
reaction mixture was continued for a total of 4.times.75 ml. of
ethyl acetate, after which the excess ethyl acetate was stripped
from the aqueous layer. An aliquot of the aqueous solution was
subjected to assay by HPLC. The resulting curve indicated a 26.4%
yield of di-Cbz BB-K8.
The aqueous layer was then hydrogenated in a Parr apparatus at 50
p.s.i. H.sub.2 pressure for two hours, using 0.5 gm. 10% Pd on
carbon catalyst. The material was filtered, and the combined
filtrate and washings were determined against E. coli to contain a
31.2% yield of BB-K8. BB-K8/BB-K29 ratio approximately 9-10/1;
traces of polyacyl and unreacted Kana A present.
EXAMPLE 8
Effect of Water on the Preparation of BB-K8 by the Acylation of
Poly(trimethylsilyl) Kana A in Acetone Solution at 23.degree.
C.
A. Anhydrous Solvent
Poly(trimethylsilyl) Kana A prepared as in Example 3 (2.40 gm., 2.0
m moles, calculated as Kana A (silyl).sub.10) was dissolved in 20
ml. of acetone which had been dried with a molecular sieve. The
solution was stirred at 23.degree. C. and a solution of SAE (0.701
gm., 2.0 m moles) in 10 ml. of sieve dried acetone was added over a
period of 10 seconds. Stirring was continued at 23.degree. C. for
22 hours. Water (50 ml.) was added and the pH (7.5) was adjusted to
2.5. The acetone was stripped in vacuo at 40.degree. C. and the
aqueous solution was then reduced at 51 p.s.i. H.sub.2 pressure at
23.degree. C. for two hours, utilizing 1.0 gm of 10% Pd on carbon
as catalyst. Microbiological assay showed a 31.24% yield of
BB-K8.
B. Water Added to Solvent
Step A, above, was repeated, except that 1.0 ml. (56 m moles) of
water was added to the poly(trimethylsilyl) Kana A solution, and
stirred for 15 minutes, prior to acylation with SAE.
Microbiological assay showed a 33.80% yield of BB-K8.
EXAMPLE 9
Preparation of BB-K8 by Acylation of Poly(trimethylsilyl) 6'-N-Cbz
Kana A in Acetone with the Mixed Anhydride of BHBA and
Isobutylcarbonic Acid
A. Preparation of Mixed Anhydride
BHBA (1,267 gm., 5.0 m moles) and N-trimethylsilylacetamide (MSA)
(1.313 gm., 10.0 m moles) in 20 ml. of sieve dried acetone was
stirred at 23.degree. C., and triethylamine (TEA) (0.70 ml., 5.0 m
moles) were added. The mixture was refluxed under a N.sub.2
atmosphere for 21/2 hours. The mixture was cooled to -20.degree. C.
and isobutylchloroformate (0.751 gm., 0-713 ml., 5.50 m moles) was
added. Triethylamine hydrochloride immediately began to separate.
The mixture was stirred for 1 hour at -20.degree. C.
B. Acylation
Poly(trimethylsilyl) 6'-N-Cbz Kana A prepared as in Example 1
(6.215 gm., 4.9 m moles, calculated as the (silyl).sub.9 compound)
was dissolved in 20 ml. of sieve dried acetone, with stirring, at
23.degree. C. The solution was cooled to -20.degree. C. and the
cold mixed anhydride solution from step A was slowly added over a
period of 30 minutes. The reaction mixture was stirred for an
additional 11/2 hours at -20.degree. C. and then for 17 hours at
23.degree. C. The reaction mixture was then poured into 150 ml. of
water at 23.degree. C. with stirring, the pH (7.75) was adjusted to
2.5 with 3 N HCl, and stirring was continued for 15 minutes.
Acetone was then stripped in vacuo at 40.degree. C. An aliquot of
the resulting aqueous solution was subjected to assay by HPLC. The
resulting curve indicated a 34.33% yield of di-Cbz BB-K8.
The main portion of the aqueous solution was reduced at 50 p.s.i.
H.sub.2 pressure at 23.degree. C. for 31/4 hours, utilizing 2.0 gms
of Pd/C catalyst. The catalyst was removed by filtration and the
combined filtrate and washings were determined by microbiological
assay against E. coli to contain a 35.0% yield of BB-K8.
EXAMPLE 10
Preparation of BB-K8 by Acylation of Poly(trimethylsilyl) 6'-N-Cbz
Kana A in 3-Pentanone
Poly(trimethylsilyl) 6'-N-Cbz Kana prepared as in Example 1 (30
gm., 23.65 m moles, calculated as 6'-N-Cbz Kana A silyl).sub.9)
dissolved in 100 ml. sieve dried 3-pentanone was stirred at
23.degree. C., and NAE (26.02 m moles, 10% excess) was added over a
period of 40 minutes. Stirring was continued for 113 hours at
23.degree. C. and the mixture was then added to 250 ml. water with
vigorous stirring. The pH (7.3) was adjusted to 2.5 with 3 N HCl,
the mixture was stirred for an additional 30 minutes, and the
3-pentanone was stripped in vacuo at 40.degree. C. The aqueous
solution was extracted with 4.times.100 ml. of ethyl acetate. An
aliquot of the aqueous solution was then subjected to assay by
HPLC. The resulting curve indicated a 46.12% yield of di-Cbz
BB-K8.
The main portion of the aqueous reaction mixture was reduced at
51.0 p.s.i. H.sub.2 pressure at 23.degree. C. for 21/2 hours,
utilizing 3.0 gms. of 10% Pd/C catalyst. Microbiological assay of
an aliquot of the combined filtrate and washings indicated a 40.24%
yield of BB-K8. The main portion of the reduced aqueous reaction
mixture was then concentrated in vacuo at 40.degree. C. to
approximately 100 ml. and fractionated on a CG-50 (NH.sub.4 +) ion
exchange column (4 inches.times.4 feet, containing approximately 10
liters of resin). The aqueous solution was charged on the column,
the column was washed with 5 liters of water, and the material was
eluted with 0.5 N NH.sub.4 OH (followed by 3 N NH.sub.4 OH to elute
polyacylated products). Polarimetry of the fractions showed the
presence of a 42.7% yield of BB-K8, a 12.0% yield of unreacted
kanamycin A, a 12.4% yield of polyacylated matrial and a 23.2%
yield of BB-K29.
EXAMPLE 11
Preparation of BB-K8 by Acylation of Poly(trimethylsilyl) 6'-N-Cbz
Kana A in Anhydrous Cyclohexanone For Varying Times
A. Poly(trimethylsilyl) 6'-N-Cbz Kana A prepared as in Example 1
(2.537 gm., 2.0 m moles, calculated as 6'-N-Cbz Kana A
(silyl).sub.9) in 300 ml. dry cyclohexanone was acylated for 20
hours at 23.degree. C. with an NAE solution in dry cyclohexanone
(10.8 ml. of 0.1944 m mole/ml. solution, 2.10 m mole). The reaction
mixture was then added to 150 ml. of water, with stirring, and the
pH (5.6) was adjusted to 2.5 with 3 N HCl. The cyclohexanone was
stripped in vacuo at 40.degree. C. and an aliquot of the remaining
aqueous phase was taken for assay by HPLC. The main portion of the
aqueous phase was reduced under 50 p.s.i. H.sub.2 pressure for 3
hours at 23.degree. C., using 1.0 gm of 10% Pd/C catalyst. The
catalyst was removed by filtration and the combined filtrate and
washings were assayed microbiologically for BB-K8.
B. Reaction A, above, was repeated, except that the acylation was
continued for 115 hours instead of 20 hours.
______________________________________ Yields Microbiological HPLC
Assay Assay (BB-K8) (di-Cbz BB-K8) Turbidimetric Plate
______________________________________ Reaction A 49.18% 42.87%
39.16% Reaction B 56.17% 55.39% 38.45%
______________________________________
EXAMPLE 12
Preparation of BB-K8 by Acylation of Poly(trimethylsilyl) 6'-N-Cbz
Kana A in Anhydrous Tetrahydrofuran For Varying Times
A. Example 11 A was repeated except that dry tetrahydrofuran was
utilized as solvent instead of dry cyclohexanone.
B. Example 11 B was repeated except that dry tetrahydrofuran was
utilized as solvent instead of dry cyclohexanone.
______________________________________ Yields Microbiological HPLC
Assay Assay (BB-K8) (di-Cbz BB-K8) Turbidimetric Plate
______________________________________ Reaction A 29.27% 28.34%
28.18% Reaction B 33.39% 21.52% 28.63%
______________________________________
EXAMPLE 13
Preparation of BB-K8 by Acylation of Poly(trimethylsilyl) 6'-N-Cbz
Kana A in Anhydrous Dioxane For Varying Times
A. Example 11 A was repeated except that the acylation was
continued for 44 hours utilizing dry dioxane as the solvent.
B. Example 11 B was repeated except that the acylation was
continued for 181/2 hours utilizing dry dioxane as the solvent.
______________________________________ Yields Microbiological HPLC
Assay Assay (BB-K8) (di-Cbz BB-K8) Turbidimetric Plate
______________________________________ Reaction A 39.18% 43.27%
33.36% Reaction B 42.82% 22.55% 33.37%
______________________________________
EXAMPLE 14
Preparation of BB-K8 by Acylation of Poly(trimethylsilyl) 6'-N-Cbz
Kana A in Anhydrous Diethyl ketone at 75.degree. C.
To a stirred solution of poly(trimethylsilyl) 6'-N-Cbz Kana A
prepared as in Example 1 (2.537 gm., 2.0 m moles, calculated as
6'-N-Cbz Kana A (silyl).sub.9) in 32 ml. sieve dried diethyl ketone
at 75.degree. C. was added a solution of NAE (10.8 ml. of 0.1944 m
moles/ml. of diethyl ketone, 2.10 m moles) over a period of 15
minutes. Stirring was continued at 75.degree. C. for an additional
3 hours after which the mixture was poured into 150 ml. of water.
The pH was adjusted to 2.8 with 3 N HCl and the diethyl ketone was
stripped in vacuo at 40.degree. C. HPLC assay of an aliquot of the
aqueous phase indicated a 39.18% yield of di-Cbz BB-K8.
The main portion of the aqueous phase was reduced under 49.8 p.s.i.
H.sub.2 pressure for 31/4 hours at 23.degree. C., using 1.0 gm of
Pd/C catalyst. The catalyst was removed by filtration and the
combined filtrate and washings were assayed microbiologically for
BB-K8. Turbidimetric assay showed 27.84% yield and Plate assay
showed 28.6% yield.
EXAMPLE 15
Preparation of BB-K8 by the Acylation of Poly(trimethylsilyl) Kana
A With NAE at 0.degree.-5.degree. After Back Hydrolysis With
Water
A. Silylation of Kanamycin A Using HMDS With TMCS as Catalyst
Kanamycin A (10 gm of 97.6% purity, 20.14 m moles) in 100 ml of
sieve-dried acetonitrile was brought to reflux under a nitrogen
atmosphere. A mixture of HMDS (22.76 gm, 141 m moles, 7 moles per
mole of kanamycin A) and TMCS (1 ml, 0.856 gm, 7.88 m moles) was
added to the refluxing reaction mixture over a period of 10
minutes. Reflux was continued for 43/4 hours and the mixture was
then cooled, concentrated in vacuo to a yellow viscous syrup and
dried under high vacuum for 2 hours. The yield of product was 23.8
gms (97.9%, calculated as kanamycin A (silyl).sub.10).
B. Acylation
Poly(trimethylsilyl) kanamycin A (23.8 gms, 20.14 m moles) prepared
in step A above was dissolved in 250 ml of sieve-dried acetone at
23.degree. and then cooled to 0.degree.-5.degree.. Water (3.63 ml,
201.4 m moles, 10 moles per mole of polysilylated kanamycin A) was
added, with stirring, and the mixture was allowed to stand under
moderate vacuum for 30 minutes, NAE (19.133 m moles, 0.95 moles per
mole of polysilylated kanamycin A) in 108.3 ml of acetone was then
added over a period of <1 minute. The mixture was stirred at
0.degree.-5.degree. for 1 hour, diluted with water, the pH adjusted
to 2.5, and the acetone was then removed in vacuo. The aqueous
solution was then reduced at 50 p.s.i. H.sub.2 pressure at
23.degree. for 21/2 hours using 2.0 gms of 10% Pd on carbon as a
catalyst. The reduced reaction mixture was filtered through
Dicalite, concentrated to ca. 100 ml in vacuo at 40.degree. and
then charged on CG-50(NH.sub.4.sup.+) column (6 liters resin,
5.times.100 cm). It was washed with water and then eluted with 0.6
N-1.0 N-3 N NH.sub.4 OH. There was obtained 60.25% BB-K8, 4.37%
BB-K6, 4.35% BB-K29, 26.47 % kanamycin A and 2.18% polyacyls.
EXAMPLE 16
Preparation of BB-K8 by the Acylation of Poly(trimethylsilyl)
6'-N-Cbz Kana A with SAE at 0.degree.-5.degree. After Back
Methanolysis
A. Silylation of 6'-N-Cbz Kanamycin A
6'-N-Cbz kanamycin A (20.0 gm, 32.4 m moles) in 200 ml of
sieve-dried acetonitrile was brought to reflux under a nitrogen
atmosphere. HMDS (47.3 ml, 226.8 m moles, 7 moles per mole of
6'-N-Cbz kana A) was added over a 10 minute period and reflux was
continued for 20 hours. The mixture was then cooled, concentrated
in vacuo, and dried under high vacuum for 2 hours to give 39.1 gms
of white amorphous solid (95.4% yield, calculated as 6'-N-Cbz kana
A (silyl).sub.9).
B. Acylation
Poly(trimethylsilyl) 6'-N-Cbz kana A (39.1 gm, 32.4 m moles)
prepared in step A above was dissolved in 400 ml of dry acetone,
with stirring, at 23.degree.. Methanol (6.6 ml, 162 m moles, 5
moles per mole of polysilylated 6'-N-Cbz kana A) was added and the
mixture was stirred at 23.degree. for 1 hour under a strong
nitrogen purge. The mixture was cooled to 0.degree.-5.degree. and a
solution of SAE (11.35 gm, 32.4 m moles) in 120 ml of pre-cooled,
dry acetone was added. The mixture was stirred for an additional 3
hours at 0.degree.-5.degree. and then placed in a 4.degree. cold
room for 1 week. Water (300 ml) was added, the pH was adjusted to
2.0, the mixture was stirred for 1 hour, and the acetone was then
stripped in vacuo. The resultant aqueous solution was reduced at
54.0 p.s.i. H.sub.2 pressure for 17 hours at 23.degree. utilizing
3.0 gm of 10% Pd on carbon as catalyst. It was then filtered
through Dicalite, concentrated in vacuo to 75-100 ml, charge on a
CG-50(NH.sub.4.sup.+) column and eluted with water and 0.6 N
NH.sub.4 OH. There was obtained 52.52% BB-K8, 14.5% BB-K29, 19.6%
kanamycin A and 1.71% polyacyls.
EXAMPLE 17
Preparation of BB-K8 by the Acylation of Poly(trimethylsilyl) Kana
A With SAE at 0.degree.-5.degree. After Back Hydrolysis With
Water
A. Silylation of Kanamycin A With TMCS in Acetonitrile Using
Tetramethylguanidine as Acid Acceptor
Kanamycin A (4.88 gm, 10.07 m mole) was suspended in 100 ml of
sieve-dried acetonitrile with stirring at 23.degree.. To the
stirred suspension was added tetramethylguanidine (TMG) (16.234 gm,
140.98 m moles, 14 moles per mole of kanamycin A). The mixture was
heated to reflux and TMCS (15.32 gm, 140.98 m moles, 14 moles per
mole of kanamycin A) was added over a 15 minute period. A white
precipitate of TMG.HCl formed after about one-half of the TMCS had
been added. The mixture was cooled to room temperature,
concentrated to a tacky residue and dried under high vacuum for 2
hours. The solid was triturated with dry THF (100 ml), and the
insoluble TMG.HCl was filtered off and washed with 5.times.20 ml
portions of THF. The combined filtrate and washings were
concentrated in vacuo at 40.degree. to a tacky residue and dried
under high vacuum for 2 hours. There was obtained 10.64 gms of a
light cream tacky residue (87.6% yield, calculated as kanamycin A
(silyl).sub.10).
B. Acylation
Poly(trimethylsilyl) kanamycin A (10.64 gm, 10.07 m moles) prepared
in step A above was dissolved in 110 ml of sieve-dried acetone,
with stirring, at 23.degree. and the solution was cooled to
0.degree.-5.degree.. Water (1.81 ml, 100.7 m moles, 10 moles per
mole of polysilylated kana A) was added and the solution was
stirred for 30 minutes under moderate vacuum. SAE (3.70 gm, 10.57 m
moles, 5% excess) in 40 ml of pre-cooled dry acetone was added over
a period of <1 minute, and the mixture was stirred for one hour.
The mixture was worked up by the general procedure in Example 16B
to give ca. 50% BB-K8, ca. 10% BB-K29, 5-8% BB-K6, ca. 20%
kanamycin A and 5-8% polyacyls.
EXAMPLE 18
Preparation of Poly(trimethylsilyl) Kanamycin A in Pyridine Using
HMDS
Kanamycin A (10.0 gms, 20.64 m moles) was suspended in 100 ml
sieve-dried freshly distilled pyridine at 23.degree.. A nitrogen
purge was started and the suspension was brought to reflux. HMDS
(17.33 gms, 107.32 m moles, 5.2 moles per mole of kanamycin A) was
added over a period of 10 minutes and the mixture was refluxed for
19 hours. It was then cooled to room temperature, concentrated in
vacuo to a light yellow-gold syrup, and dried under high vacuum to
a white amorphous solid. There was obtained 22.1 gms (92.6% yield,
calculated as kanamycin A (silyl).sub.10).
EXAMPLE 19
Preparation of Poly(triethylsilyl) Kanamycin A Using
Triethylchlorosilane With Triethylamine as Acid Acceptor
Kanamycin A (5.0 gms of 97.6% purity, 10.07 m moles) was suspended
in 100 ml of sieve-dried acetonitrile at 23.degree.. Triethylamine
(TEA) (33.8 ml, 24.5 gm, 241.7 m moles) was added and the
suspension was brought to reflux. A solution of
trichloroethylsilane (23.7 ml, 21.3 gm, 140.98 m moles) in 25 ml
dry acetonitrile was added over a 20 minute period. Reflux was
continued for an additional 7 hours and the mixture was cooled to
room temperature, whereupon long fine needles of TEA.HCl separated
out. The mixture was allowed to stand at room temperature for ca.
16 hours, concentrated in vacuo at 40.degree. to a tacky solid and
dried for 2 hours under high vacuum to a deep orange tacky solid.
The solid was triturated with 100 ml dry THF at 23.degree. and the
insoluble TEA.HCl was filtered off, washed with 5.times.20 ml of
THF, and dried to give 16.0 gms of TEA.HCl. The combined filtrate
and washings were concentrated in vacuo to a solid and dried under
high vacuum for 2 hours. There was obtained 19.3 gms of
poly(triethylsilyl) kanamycin A as a deep orange viscous syrup.
EXAMPLE 20
Preparation of Poly(trimethylsilyl) Kanamycin A Using
bis-Trimethylsilylurea
Kanamycin A (10.0 gm of 99.7% purity, 20.58 m moles) was suspended
in 200 ml of sieve-dried acetonitrile, with stirring, at
23.degree.. To the suspension was added bis-trimethylsilylurea
(BSU) (29.45 gms, 144.01 m moles, 7 moles per mole of kanamycin),
and the mixture was brought to reflux under a nitrogen atmosphere.
Reflux was continued for 17 hours and the reaction mixture was then
cooled to room temperature. A small amount of insoluble material
present was removed by filtration, washed with 3.times.10 ml
portions of acetonitrile and dried (1.1381 gms). Infrared showed
this to be BSU plus a small amount of unreacted kanamycin A. The
combined filtrate and washings were cooled at 4.degree. for 16
hours. Additional solid separated, was recovered as above, (7.8
gms) and was shown by infrared to be BSU plus urea. The light
yellow filtrate and washings were concentrated in vacuo at
40.degree. and dried under high vacuum to give 27.0 gm of a white
solid which was partly tacky and partially fine needle-like
crystals. The solid was treated with 150 ml of heptane at
23.degree., the insoluble portion was removed by filtration, washed
with 2.times.50 ml portions of heptane and dried, to give 6.0 gms
of white needles (shown by infrared to be BSU plus urea). The
combined filtrate and washings were concentrated in vacuo at
40.degree. and dried under high vacuum for 2 hours to give 20.4 gms
of white needles, the infrared spectrum of which was typical for
polysilylated kanamycin A. Calculations showed the product to
contain an average of 7.22 trimethylsilyl groups.
EXAMPLE 21
Preparation of BB-K8 by the Acylation of Per(trimethylsilyl)
Kanamycin A After Partial Desilylation With 1,3-Butanediol
A. Preparation of Per(trimethylsilyl) kanamycin A
Kanamycin A (10.0 gm, 20.639 m moles) was suspended in 100 ml of
sieve-dried acetonitrile, with stirring, at 23.degree.. The
suspension was brought to reflux under a nitrogen purge and HMDS
(23.322 gms, 144.5 m moles, 7 moles per mole of kanamycin A) was
added over a period of ten minutes. Reflux was continued for 16
hours and the mixture was then cooled to room temperature,
concentrated in vacuo and dried for 2 hours under high vacuum.
There was obtained 24.3 gm of a white, tacky residue (92.1% yield,
calculated as kanamycin A (silyl).sub.11).
B. Acylation
Per(trimethylsilyl) kanamycin A (24.3 gm) prepared in step A above
was dissolved in 240 ml of sieve-dried acetone, with stirring, at
23.degree.. To this solution was added 1,3-butanediol (9.25 ml,
103.2 m mole, 5 moles per mole of per(trimethylsilyl) kanamycin A.
The mixture was stirred at 23.degree. for 2 hours under a nitrogen
purge and then cooled at 0.degree.-5.degree.. SAE (7.23 gm, 20.64 m
moles) in 70 ml of pre-cooled acetone was added over a period of
about 1 minute. The mixture was stirred at 0.degree.-5.degree. for
3 hours and then allowed to stand in a 4.degree. cold room for ca.
16 hours. Water (200 ml) was added, the pH was adjusted to 2.5 and
the clear yellow solution was stirred at 23.degree. for 30 minutes.
The acetone was stripped in vacuo and the aqueous solution was
reduced at 55.0 p.s.i. H.sub.2 pressure at 23.degree. for 2 hours
using 3.0 gm of 10% Pd on carbon as catalyst. The reduced solution
was filtered through Dicalite and chromatographed as in Example 16B
to give 47.50% BB-K8, 5.87% BB-K29, 7.32% BB-K6, 24.26% kanamycin A
and 7.41% polyacyls.
EXAMPLE 22
Preparation of BB-K8 by the Acylation of Poly(trimethylsilyl)
Kanamycin A Prepared in THF Using SAE With Sulfamic Acid
Catalyst
To a refluxing mixture of kanamycin A (5.0 gm, 10.32 m moles) in 50
ml of sieve-dried tetrahydrofuran (THF) were added sulfamic acid
(100 mg) and HMDS (12.32 gm, 76.33 m moles). The mixture was
refluxed for 18 hours, with complete solution occurring after 6
hours. The solution was cooled to 23.degree., treated with 0.1 ml
of water and held at 23.degree. for 30 minutes. After stirring for
3 hours, the mixture was diluted with 100 ml of water and the pH
was adjusted to 2.2 with 10% H.sub.2 SO.sub.4. It was stirred for
30 minutes at 23.degree. and then concentrated in vacuo to remove
THF. The resulting aqueous solution was reduced at 50 p.s.i.
H.sub.2 pressure for 2 hours at 23.degree. using 10% Pd on carbon
as a catalyst. The reduced solution was filtered through Dicalite
and the solids were washed with water. The combined filtrate and
washings (150 ml) were determined by microbiological assay against
E. coli to contain 1225 mcg/ml (31.5% activity yield) of BB-K8.
EXAMPLE 23
Preparation of BB-K8 by the Acylation of Poly(trimethylsilyl)
Kanamycin A with the N-Hydroxysuccinimide Ester of
Di-Carbobenzyloxy AHBA
A. Preparation of Dicarbobenzyloxy
L-(-)-.alpha.-Amino-.alpha.-hydroxybutyric Acid
N-Hydroxysuccinimide Ester
Dicarbobenzyloxy L-(-)-.alpha.-amino-.alpha.-hydroxybutyric acid (8
gm, 20.65 m moles) and N-hydroxysuccinimide (2.37 gm, 20.65 m
moles) were dissolved in 50 ml of dry acetone at 23.degree..
Dicyclohexylcarbodiimide (4.25 gm, 20.65 m moles) dissolved in 20
ml of dry acetone was added and the total was agitated at
23.degree. for 2 hours. Dicyclohexylurea was filtered off, the
filter cake was washed with 10 ml of dry acetone, and the filtrate
and washings were combined.
B. Acylation
Poly(trimethylsilyl) kanamycin A, prepared according to the general
procedure of Example 21 from 10.0 gms (20.639 m moles) of kanamycin
A, was dissolved in 100 ml of dry acetone. The solution was cooled
to 0.degree.-5.degree., 3.7 ml of deionized water was added, and
the solution was stirred at 0.degree.-5.degree. for 30 minutes
under moderate vacuum.
To this solution was added the solution of the di-Cbz-blocked
acylating agent prepared in step A, and the mixture was stirred at
0.degree.14 5.degree. for 30 minutes. The mixture was diluted with
water, the pH was adjusted to 2.2 and the acetone was removed in
vacuo. The aqueous solution was reduced by the general procedure of
Example 22 and then filtered through Dicalite. Chromatography
showed 40-45% BB-K8, ca. 10% BB-K29, a trace of BB-K6, ca. 30%
kanamycin A and a small amount of polyacyls.
EXAMPLE 24
Preparation of Poly(trimethylsilyl) Kanamycin A Using HMDS with
Imidazole as Catalyst
Kanamycin A (11 gm, 22.7 m moles) and 100 mg of imidazole were
heated to reflux in 100 ml of sieve-dried acetonitrile, under a
nitrogen purge. HMDS (18.48 gm, 114.5 m moles, 5 moles per mole of
kanamycin A) was added over a period of 30 minutes and the mixture
was refluxed for 20 hours. Complete solution occurred in ca. 21/2
hours. The solution was cooled to 23.degree. and the solvent was
removed in vacuo to leave 21.6 gms of poly(trimethylsilyl)
kanamycin A as a foamy residue (93.1% yield, calculated as
kanamycin(silyl).sub.11).
EXAMPLE 25
Preparation of
1-N-[L-(-)-.gamma.-Amino-.alpha.-hydroxybutyryl]kanamycin B
(BB-K26) by the Acylation of Poly(trimethylsilyl) Kanamycin B With
SAE
A. Preparation of Poly(trimethylsilyl) Kanamycin B Using HMDS With
TMCS Catalyst
Kanamycin B (25 gm, 51.7 m moles) in 250 ml of sieve-dried
acetonitrile was heated to reflux under a stream of nitrogen. HMDS
(62.3 gm, 385.81 m moles, 7.5 moles per mole of kanamycin B) was
added over a period of 30 minutes followed by 1 ml of TMCS as
catalyst. The mixture was refluxed for 21 hours with complete
solution after 1 hour. The solvent was then removed in vacuo at
60.degree. and the oily residue was held at 60.degree. under high
vacuum for 3 hours. There was obtained 53.0 gm of
poly(trimethylsilyl) kanamycin B (85.2% yield, calculated as
kanamycin B (silyl).sub.10).
B. Acylation
The poly(trimethylsilyl) kanamycin B prepared in step A above (53.0
gm) was dissolved in 500 ml of dry acetone at 0.degree.-5.degree.,
methanol (20.9 ml) was added, and the mixture was stirred in vacuo
for 30 minutes at 0.degree.-5.degree.. A solution of SAE (18.1 gm,
51.67 m moles) in 200 ml of pre-cooled dry acetone was added over a
period of less than 1 minute and the mixture was stirred for 30
minutes at 0.degree.-5.degree.. The mixture was worked up according
to the general procedure of Example 22 and then loaded on a column
of CG-50 (NH.sub.4.sup.+) (8.times.120 cm). It was eluted with an
NH.sub.4 OH gradient from 0.6 N to 3 N. There was obtained 38% of
BB-K26, 5% of the corresponding 6'-N-acylated kanamycin B (BB-K22),
10% of the corresponding 3-N-acylated kanamycin B (BB-K46) 14.63%
kanamycin B and a small amount of polyacylated kanamycin B.
EXAMPLE 26
Preparation of Poly(trimethylsilyl) Kanamycin A Using HMDS With
Kanamycin A Sulfate as Catalyst
Kanamycin A (19.5 gm, 40.246 m moles) and kanamycin A sulfate (0.5
gm, 0.858 m mole) [total=20.0 gm, 41.0 m moles] in 200 ml of
sieve-dried acetonitrile was brought to reflux. HMDS (60.3 ml,
287.7 m moles, 7 moles per mole of kanamycin A) was slowly added
and the mixture was refluxed for 28 hours. It was then stripped to
dryness on a rotary evaporator and dried under steam injector
vacuum. There was obtained 47.5 gms of poly(trimethylsilyl)
kanamycin A as a pale yellow oil (95.82% yield, calculated as
kanamycin A (silyl).sub.10).
EXAMPLE 27
Preparation of BB-K8 by the Acylation of Poly(trimethylsilyl)
Kanamycin A With N-Trifluoroacetyl Blocked AHBA
N-Hydroxysuccinimide Ester
A. Preparation of N-Trifluoroacetyl AHBA and Conversion to its
N-Hydroxysuccinimide Ester
To a suspension of AHBA (5.0 gm, 42 m moles) in 100 ml THF was
added trifluoroacetic anhydride (40 gm, 191 m moles), with
stirring, over a 10 minute period. The solution was stirred for 18
hours at 23.degree. and then concentrated to dryness in vacuo at
50.degree.. The residue was dissolved in 100 ml of aqueous methanol
(1:1) and stirred for 1 hour. It was then concentrated to dryness
in vacuo and redissolved in 50 ml H.sub.2 O. The aqueous solution
was extracted with 3.times.50 ml portions of MIBK and, after drying
over Na.sub.2 SO.sub.4, the extract was concentrated to an oil.
Traces of solvent were removed by adding and distilling off 4 ml of
water. On standing the oil changed to a waxy, crystalline solid
(2.5 gm, 28% yield.
The N-trifluoroacetyl AHBA (2.4 gm, 11.3 m moles) was dissolved in
50 ml dry acetone and N-hydroxysuccinimide (1.30 gm, 11.31 m moles)
was added to the solution. A solution of dicyclohexylcarbodiimide
(2.33 gm) in 20 ml of dry acetone was slowly added. The reaction
mixture was stirred for 2 hours at 23.degree. and the precipitated
dicyclohexylurea was removed by filtration and washed with a small
amount of acetone. The combined filtrate and washings (a solution
of the N-hydroxysuccinimide ester of N-trifluoroacetyl AHBA) was
utilized in the next step without isolation.
B. Acylation
To a solution of poly(trimethylsilyl) kanamycin A prepared as in
Example 26 (11.31 m moles) in 54 ml of acetone was added 2.0 ml
(113.4 m moles) of water, and the mixture was stirred in vacuo at
0.degree.-5.degree. for 30 minutes. The N-hydroxysuccinimide ester
of N-trifluoroacetyl AHBA prepared in step A above (11.31 m moles)
was added to the mixture and it was then held at 5.degree. for 1
hour. The pH was then adjusted to ca. 2.0 with 20% H.sub.2
SO.sub.4, the mixture was stirred for 30 minutes and the pH was
then raised to ca. 6.0 with NH.sub.4 OH. The mixture was then
stripped to dryness in a rotary evaporator to give 14.4 gm of a
tacky off-white solid. The solid was dissolved in 100 ml of water,
the pH was raised from 5.5 to 11.0 with 10 N NH.sub.4 OH and the
solution was heated in an oil bath at 70.degree. for 1 hour. The pH
(9.5) was then lowered to 7.0 with HCl, the solution was polish
filtered to remove a small amount of insolubles and the filter was
washed with water. The combined filtrate and washings (188 ml) was
applied to a CG-50 (NH.sub.4.sup.+) column (8.times.90 cm), washed
with 2 liters of water and eluted with a NH.sub.4 OH gradient (0.6
N-1.0 N-concentrated). There was obtained 28.9% BB-K8, 5.0% BB-K6,
5.7% BB-K29, 43.8% kanamycin A, 3.25% polyacyls plus 14.3% of an
unknown material which was in the first fraction off the
column.
EXAMPLE 28
Preparation of BB-K8 by the Acylation of Poly(trimethylsilyl)
Kanamycin A With t-Butyloxycarbonyl Blocked AHBA
N-Hydroxysuccinimide Ester
A. Preparation of t-BOC AHBA and Conversion to its
N-Hydroxysuccinimide Ester
A solution of AHBA (5.0 gm, 42 m moles) in 100 ml of water and 20
ml of acetone was adjusted to pH 10 with 10 N NaOH. Over a period
of 3-4 minutes was added 11.6 gm (53 m moles) of di-t-butyl
dicarbonate, and the solution was stirred for 35 minutes while
maintaining the pH at 10 by the periodic addition of 10 N NaOH. The
acetone was removed in vacuo and the aqueous phase was washed with
40 ml of ethyl acetate. The pH of the aqueous solution was lowered
to 2.0 with 3 N HCl and it was then extracted with 3.times.30 ml of
MIBK. The combined MIBK extracts were dried over Na.sub.2 SO.sub.4
and concentrated to a clear oily residue (8.2 gm, 89%).
The t-BOC-AHBA (4.25 gm, 19.4 m moles) was dissolved in 50 ml of
acetone and N-hydroxysuccinimide (2.23 gm, 19.4 m moles) was added.
A solution of dicyclohexylcarbodiimide (4.00 gm 19.4 m moles) in 20
ml of acetone was slowly added and the mixture was stirred for 2
hours at 23.degree.. The precipitated dicyclohexylurea was removed
by filtration and was washed with a small amount of acetone. The
combined filtrate and washings (a solution of the
N-hydroxysuccinimide ester of t-BOC-AHBA) was utilized in the next
step without isolation.
B. Acylation
To a solution of poly(trimethylsilyl) kanamycin A prepared as in
Example 26 (41.28 m moles) in 94 ml of acetone was added 3.5 ml
(194 m moles) of water, and the mixture was stirred in vacuo at
0.degree.-5.degree. for 30 minutes. The N-hydroxysuccinimide ester
of t-BOC-AHBA prepared in step A above (19.4 m moles) was added and
the mixture was allowed to stand at 5.degree. for 1 hour. Water
(200 ml) was added and the pH (7.0) was lowered to 2.0 with 20%
H.sub.2 SO.sub.4. After 30 minutes stirring the pH was raised to
ca. 6.0 with NH.sub.4 OH and the mixture was stripped to dryness in
vacuo to give 36.3 gms of a golden oil. The oil was dissolved in
200 ml of trifluoroacetic acid, allowed to stand for 15 minutes and
stripped to dryness in a rotary evaporator. The oil was washed with
water and the water was flashed off. Concentrated NH.sub.4 OH was
added to pH 6.0 and was flashed off. The resulting solid was
dissolved in water, filtered, and the filter washed with water. The
combined filtrate and washings (259 ml) were loaded on a CG-50
(NH.sub.4.sup.+) column (8.times.92 cm), washed with 4 liters of
water and eluted with an NH.sub.4 OH gradient (0.6 N-1.0
N-concentrated). There was obtained 40.32% BB-K8, 4.58% BB-K6,
8.32% BB-K29, 30.50% kanamycin A and 7.43% polyacyls.
EXAMPLE 29
The general procedure of Example 1 is repeated, except that the
6'-N-carbobenzyloxykanamycin A used therein is replaced by an
equimolar weight of 6'-N-carbobenzyloxykanamycin B, and there is
thereby produced
1-N-[L-(-)-.gamma.-amino-.alpha.-hydroxybutyryl]kanamycin B.
EXAMPLE 30
The general procedure of Example 1 is repeated except that the
L-(-)-.gamma.-benzyloxycarbonylamino-.alpha.-hydroxybutyric acid
N-hydroxy-5-norbornene-2,3-dicarboximide ester used therein is
replaced by
L-(-)-.beta.-benzyloxycarbonylamino-.alpha.-hydroxypropionic acid
N-hydroxy-5-norbornene-2,3-dicarboximide ester and
L-(-)-.delta.-benzyloxycarbonylamino-.alpha.-hydroxyvaleric acid
N-hydroxy-5-norbornene-2,3-dicarboximide ester, respectively,
and there is thereby produced
1-N-[L-(-)-.beta.-amino-.alpha.-hydroxypropionyl]kanamycin A
and
1-N-[L-(-)-.delta.-amino-.alpha.-hydroxyvaleryl]kanamycin A,
respectively.
EXAMPLE 31
The general procedure of Example 25 is repeated except that the
L-(-)-.gamma.-benzyloxycarbonylamino-.alpha.-hydroxybutyric acid
N-hydroxyester used therein is replaced by
L-(-)-.beta.-benzyloxycarbonylamino-.alpha.-hydroxy propionic acid
N-hydroxysuccinimide ester and
L-(-)-.delta.-benzyloxycarbonylamino-.alpha.-hydroxyvaleric acid
N-hydroxysuccinimide ester, respectively,
and there is thereby produced
1-N-[L-(-)-.beta.-amino-.alpha.-hydroxypropionyl]kanamycin B
and
1-N-[L-(-)-.delta.-amino-.alpha.-hydroxyvaleryl]kanamycin B,
respectively.
EXAMPLE 32
Preparation of BB-K8 by the Acylation of Poly(trimethylsilyl)
3,6'-Di-N-Carbobenzyloxykanamycin A in Anhydrous Diethyl Ketone
A. 3,6'-Di-N-carbobenzyloxykanamycin A
A suspension of 7.26 g (15 m moles) of kanamycin A (free base) and
18.6 g (75 m moles) of nickel acetate tetrahydrate in 300 ml of
dimethylsulfoxide (DMSO) was heated at 100.degree. C. for about 30
minutes, with stirring, until a clear, green solution was obtained.
After cooling, a solution of 11.8 g (37.6 m moles) of
N-carbobenzyloxy-5-norbornene-2,3-dicarboximide in 50 ml of DMSO
was added to the solution. The mixture was stirred at room
temperature overnight, treated with 100 ml of concentrated ammonia
water and 1 liter of water, stirred at room temperature for 1 hour,
and applied to the top of a Diaion HP-10 column (300 ml). The
column was subjected to stepwise elution beginning with 7 N
NH.sub.4 OH, then with methanol-water (1:1) and finally with
methanol-water (10:1), collecting 20 ml fractions and monitoring by
thin layer chromatography on Merck Silica Gel 60 F-254 plates using
chloroform-ethanol-28% ammonium hydroxide (1:2:1). A portion of the
desired product which crystallized as fine needles from fractions
containing the product at high concentrations was filtered off to
provide an analytical sample. The filtrate and other fractions
containing the desired product (RF 0.42) were combined, and the
solution was evaporated in vacuo. The residue was triturated with
diethyl ether to give a total of 9.7 g (86%) of the title product.
Mp >300.degree. C. IR(KBr): .nu.c=o 1690 cm.sup.-1.
NMR(DMSO-d.sub.6 +DCl, pD ca 3): .delta.4.76-5.26 (6H, m, H.sub.1
',H.sub.1 " and CO-OCH.sub.2 -C.sub.6 H.sub.5 .times.2), 7.26(10H,
s, CO-OCH.sub.2 -C.sub.6 H.sub.5 .times.2).
Anal. Calcd. for C.sub.34 H.sub.48 N.sub.4 O.sub.15.H.sub.2 O: C,
52.98; H, 6.54; N, 7.27. Found: C, 53.20; H, 6.42; N, 7.04.
B. Poly(trimethylsilyl) 3,6'-Di-N-carbobenzyloxykanamycin A
A mixture of 1.5 g (2 m moles) of 3,6'-di-N-carbobenzyloxykanamycin
A from Step A, above, and 1.29 g (8 m moles) of
hexamethyldisilazane in 15 ml of dry acetonitrile was refluxed for
16 hours. The clear solution was concentrated to dryness in vacuo
and the residue was dissolved in 20 ml of dry diethyl ketone. The
solution was used directly in the next step.
C. Acylation of Poly(trimethylsilyl)
3,6'-Di-N-carbobenzyloxykanamycin A Utilizing an Equimolar Amount
of Acylating Agent
To the solution from Step B, above, was added, with stirring, 700
mg (2 m moles) of
L-(-)-.alpha.-carbobenzyloxyamino-.alpha.-hydroxybutyric acid
N-hydroxysuccinimide ester (SAE). The mixture was stirred at room
temperature for 19 hours, then treated with 8 ml of water and 35 ml
of tetrahydrofuran (THF), adjusted to pH 3 with aqueous
hydrochloric acid, stirred for 30 minutes and concentrated to
dryness in vacuo. The residue was dissolved in a mixture of 30 ml
of water, 40 ml of methanol, 10 ml of n-butanol and 40 ml of THF,
and hydrogenated overnight in the presence of 500 mg of 10%
palladium on carbon. The catalyst was removed by filtration and the
filtrate was evaporated in vacuo and lyophilized to give 1.7 g of
crude BB-K8. The amorphous powder was redissolved in water, and the
solution was adjusted to pH 4 with aqueous hydrochloric acid and
chromatographed on a column of Amberlite CG-50 in the
NH.sub.4.sup.+ cycle. The column was subjected to stepwise elution
with water, 0.1 N NH.sub.4 OH, 0.3 N NH.sub.4 OH, 0.5 N NH.sub.4 OH
and 2 N NH.sub.4 OH, collecting 10-ml fractions and monitoring by
thin layer chromatograph using Merck Silica Gel 60 F-254 plates
using chloroform-methanol-28% ammonium hydroxide-water (1:4:2:1).
The homogeneous fractions were combined, evaoporated and finally
lyophilized. Fractions containing BB-K8 and fractions containing
recovered kanamycin A were assayed using K. pneumoniae A20680 and
B. subtilis PCI 129, respectively.
______________________________________ Frac- tion NH.sub.4 OH Yield
Rf Nos. (N) Weight (%) Product value
______________________________________ 32-39 0.1, 0.3 102 mg
partially deblocked product 40-46 0.3 174 mg.sup.a 7.sup.c
kanamycin A 0.42 47-59 0.3, 0.5 106 mg unidentified 0.33 product
60-78 0.5 816 mg.sup.b 67.sup.c BB-K8 0.18 89-95 2 70 mg 5 diacyl-
0.05 kanamycin A ______________________________________ .sup.a 408
mcg/mg. .sup.b 956 mcg/mg. .sup.c based on bioassay.
Hydrogenolysis of the partially deblocked product with Pd-C
followed by isolation on an CG-50 column gave an additional 30 mg
(2%) of BB-K8. The total yield of BB-K8 amounted to 846 mg
(69%).
D. Acylation of Poly(trimethylsilyl)
3,6'-Di-N-carbobenzyloxykanamycin A Utilizing 1.2 Equivalents of
Acylating Agent
Step C, above, was repeated except that 20% excess acylating agent
was utilized. The following results were obtained.
______________________________________ Frac- tion NH.sub.4 OH Yield
Rf Nos. (N) Weight (%) Product value
______________________________________ 28-29 0.2 107 mg partially
deblocked product 30-41 0.2 157 mg unidentified 0.35 product 42-52
0.3 120 mg unidentified 0.30 product 53-81 0.3, 0.5 .sup. 750
mg.sup.a .sup. 60.sup.b BB-K8 0.14 94-116 1, 2 147 mg 11 diacyl-
0.05 kanamycin A ______________________________________ .sup.a 933
mcg/mg. .sup.b based on bioassay.
Hydrogenolysis of the partially deblocked product with Pd-C
followed by isolation on an CG-50 column gave an additional 21 mg
(2%) of BB-K8. The total yield of BB-K8 amounted to 771 mg
(62%).
E. Acylation of Poly(trimethylsilyl)
3,6'-Di-N-carbobenzyloxykanamycin A Utilizing 1.5 Equivalents of
Acylating Agent
Step C, above, was repeated except that 50% excess acylating agent
was utilized. The following results were obtained.
______________________________________ Rf value Frac- (twice- tion
NH.sub.4 OH Yield devel- Nos. (N) Weight (%) Product oped)
______________________________________ 29-39 0.2 293 mg partially
deblocked product 40-47 0.3 95 mg.sup.a 6.sup.c kanamycin A 0.67
65-80 0.5 582 mg.sup.b 29.sup.c BB-K8 0.33 100-130 1, 2 543 mg 26
diacyl- 0.09 kanamycin A ______________________________________
.sup.a 860 mcg/mg. .sup.b 880 mcg/mg. .sup.c based on bioassay.
* * * * *